Minimal saponin analogues, synthesis and use thereof

ABSTRACT

Truncated triterpene saponin analogues containing a trisaccharide or tetrasaccharide ester are disclosed. Also disclosed are pharmaceutical compositions comprising truncated saponin analogues and synthetic methods of producing the truncated saponin analogues. Another aspect of the present application relates to a method for immunizing a subject, comprising administering to the subject the pharmaceutical composition comprising a minimal saponin analogue and an antigen.

CROSS-REFERENCE TO PRIOR APPLICATIONS

This application claims priority of U.S. Provisional Application No.62/005,302, filed on May 30, 2014.

FIELD

The present disclosure relates generally to triterpene glycosidesaponin-derived adjuvants, syntheses thereof, and intermediates thereto.The invention also provides pharmaceutical compositions comprisingcompounds of the present invention and methods of using said compoundsor compositions in the treatment of infectious diseases and cancer.

BACKGROUND

The clinical success of anticancer and antimicrobial vaccines criticallydepends on the identification of, and access to, novel potent adjuvantswith attenuated toxicity. Molecular vaccines comprised of subunitantigens are often less immunogenic than whole pathogens and do notelicit adequate immune responses alone, requiring the inclusion of animmunoadjuvant to increase immunogenicity. Unfortunately, few adjuvantsare sufficiently potent and non-toxic for clinical use. In this context,specific fractions from extracts of the bark of Quillaja saponaria (QS)have proven to be exceedingly powerful adjuvants in immunotherapy.QS-21, a saponin natural product from the Quillaja saponaria tree, isone of the most promising adjuvants currently under investigation (FIG.1a ). It is composed of two isomeric constituents, QS-21-apiose (1a) andQS-21-xylose (1b), which differ at the terminal sugar in the lineartetrasaccharide domain. QS-21 has emerged as the immunopotentiator ofchoice in many recent clinical trials and vaccines containing QS-21 areunder development for several cancers and infectious andneurodegenerative diseases (malaria, HIV, hepatitis, tuberculosis,Alzheimer's disease). Despite its promise, QS-21 suffers from severalliabilities including limited access from its natural source, toxic sideeffects, and chemical instability due to spontaneous hydrolysis of theacyl chain. Furthermore, poor understanding of its molecular mechanismof action impedes rational development of analogues with improvedefficacy and decreased toxicity.

SUMMARY

One aspect of the present application relates to a minimal saponinanalogue having the structure of formula (I),

or a pharmaceutically acceptable salt thereof, wherein

is a single or double bond; W is C(O)R, CH₂OR or CH₂R, wherein R is H,or an optionally substituted group selected from acyl, arylalkyl, aryl,heteroaryl, aliphatic, heteroaliphatic, cycloaliphatic and heterocyclylgroups; V is H or OH; Y is O; Z is a linear oligosaccharide or anoptionally substituted group selected from the group consisting ofamine, amide, acyl, arylalkyl, aryl, heteroaryl, aliphatic,heteroaliphatic, cycloaliphatic and heterocyclyl groups.

Another aspect of the present application relates to a minimal saponinanalogue having the structure of formula (II),

or a pharmaceutically acceptable salt thereof, wherein W is C(O)R, CH₂ORor CH₂R, wherein R is H, or an optionally substituted group selectedfrom acyl, arylalkyl, aryl, heteroaryl, aliphatic, heteroaliphatic,cycloaliphatic and heterocyclyl groups; V is H or OH; X is CH₂R_(m),C(O)R_(m), CH₂OR_(m), CH₂R_(m), OR_(m), or NHR_(m), wherein R_(m) is H,or an optionally substituted group selected from acyl, arylalkyl, aryl,heteroaryl, aliphatic, heteroaliphatic, cycloaliphatic and heterocyclyigroups, and each occurrence of R_(n) is independently a hydrogen, amonosaccharide, a disaccharide or a trisaccharide.

Another aspect of the present application relates to a pharmaceuticalcomposition comprising a minimal saponin analogue of the presentapplication, or a pharmaceutically acceptable salt thereof; and animmunologically effective amount of an antigen.

Another aspect of the present application relates to a method forimmunizing a subject, comprising administering to the subject thepharmaceutical composition comprising a minimal saponin analogue and anantigen.

Another aspect of the present application relates to a method forimmunizing a subject with an antigen, comprising: administering to thesubject a vaccine comprising: an effective amount of the antigen; and aneffective amount of the compound of formula (I) or a pharmaceuticallyacceptable salt thereof. In some embodiments, the vaccine isadministered orally. In other embodiments, the vaccine is administeredintramuscularly. In other embodiments, the vaccine is administeredsubcutaneously. In certain embodiments, the amount of the compound offormula (I) administered is 10-1000 μg, 500-1000 μg, 100-500 μg, 50-250μg, 50-500 μg or 250-500 μg.

Another aspect of the present application relates to a pharmaceuticalcomposition, comprising a minimal saponin analogue of the presentapplication, or a pharmaceutically acceptable salt thereof, and aneffective amount of a cytotoxic drug.

Another aspect of the present application relates to a method forenhancing the effect of a cytotoxic drug in a subject, comprisingadministering to the subject the pharmaceutical composition comprising aminimal saponin analogue of the present application and a cytotoxicdrug.

Another aspect of the present application relates to a method forenhancing the effect of a cytotoxic drug in a subject, comprising:administering to the subject a pharmaceutical composition comprising:the cytotoxic drug; and an effective amount of the compound of formula(I) or a pharmaceutically acceptable salt thereof.

Another aspect of the present application relates to a kit comprisingthe minimal saponin analogues of the present application. In someembodiments, the kits comprise prescribing information. In someembodiments, such kits include the combination of an inventive adjuvantcompound and another immunotherapeutic agent. The agents may be packagedseparately or together. The kit optionally includes instructions forprescribing the medication. In certain embodiments, the kit includesmultiple doses of each agent. The kit may include sufficient quantitiesof each component to treat a subject for a week, two weeks, three weeks,four weeks, or multiple months. In certain embodiments, the kit includesone cycle of immunotherapy. In certain embodiments, the kit includessufficient quantity of a pharmaceutical composition to immunize asubject against an antigen long term.

Another aspect of the present application relates to a compound offormula (III),

wherein W is Me, —CHO, or —CH₂OH, and V is H or OH.

Another aspect of the present application relates to a process forpreparing the compound of formula (III).

The accompanying drawings illustrate one or more embodiments of thepresent disclosure and, together with the written description, serve toexplain the principles of the exemplary embodiments of the presentdisclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings illustrate one or more embodiments of thepresent disclosure and, together with the written description, serve toexplain the principles of the exemplary embodiments of the presentdisclosure.

FIG. 1 illustrates aryl iodide saponin 6 exhibits potent adjuvantactivity and low toxicity in a preclinical mouse vaccination model.(Panel a) Structure of QS-21 and its four key structural domains. (Panelb) Synthesis of aryl iodide saponins 6 (SQS-0-0-5-18) and [131I]-6: (i)FITC, Et3N, DMF, 21° C., 2 h, 75%; (ii) 4, Et₃N, DMF, 21° C., 1 h, 52%;(iii) 5, Et₃N, DMF, 21° C., 1 h, 75%; (iv) [131I]-NaI, Chloramine-T,MeOH, 21° C., 1 min, >50%. (Panel c) Structure of adjuvant-attenuatednegative control saponin 8 (SQS-0-3-7-18) and synthesis of [131I] 8: (v)[131I]-NaI, Chloramine-T, MeOH, 21° C., 1 min, >50%. Biologicalevaluation of aryl iodide saponin 6 (SQS-0-0-5-18) with three-componentvaccine for (Panel d) anti-KLH titers (IgG), (Panel e) anti-MUC1 titers(IgG), and (Panel f) anti-OVA titers (IgG) indicating potent adjuvantactivity comparable to natural and synthetic QS-21 (compare 20 g doses);horizontal bars indicate median titers; statistical significancecompared to no-adjuvant control: *=p≦0.05, **=p<0.01, ***=p<0.001.(Panel g) Toxicity assessment based on median percent weight loss,indicating low toxicity of 6 (SQS-0-0-5-18); error bars indicate maximumand minimum values for five mice.

FIG. 2 illustrates various radioiodinated saponin [131I]-6 andfluorescent saponin 3 localize to lymph nodes and injection site inmice. (Panel a) Biodistribution of active adjuvant [131I]-6([131I]-SQS-0-0-5-18) and attenuated adjuvant [131I]-8([131I]-SQS-0-3-7-18) with OVA antigen, indicating accumulation of[131I] 6, but not [131I]-8, at injection site and lymph nodes; errorbars indicate standard deviation from mean for five mice; statisticalsignificance indicated graphically only for lymph nodes and injectionsite for clarity: *=p≦0.05: liver, muscle, lymph node, skin, thyroid;**=p<0.01: blood, lungs, spleen, kidneys, bone, injection site;***=p<0.001: heart. (Panel b) Imaging at injection site (yellow arrowsindicate ink circles) with fluorescein-labeled active adjuvant 3(SQS-0-0-5-12) or unlabeled inactive adjuvant 2 (SQS-0-0-5-11) andAlexa-647-labeled OVA (OVA-A647), indicating retention of 3 and OVA-A647at injection site; green crescent in fluorescein image for Mouse 2 isdue to software ghosting effect. (Panel c) Imaging of dissected lymphnodes with active adjuvant 3 (SQS-0-0-5-12) or inactive adjuvant 2(SQS-0-0-5-11) and OVA-A647, indicating increased accumulation ofOVA-A647 with 3 but not 2. Mice were injected in left flank and rightlymph node serves as negative control within each animal.

FIG. 3 illustrates truncated saponin 16 lacks the entire branchedtrisaccharide domain of QS-21 but retains potent adjuvant activity andlow toxicity in a preclinical mouse vaccination model. (Panel a)Synthesis of aryl iodide saponins 16 (SQS-1-0-5-18) and [¹³¹I]⁻¹⁶. (i)TESOTf, 2,6-lutidine, CH₂Cl₂, 0° C., 1 h, 80%; (ii) 1. 12, BF3.OEt2, 4 ÅM.S., CH₂Cl₂, −35° C., 30 min; 2. PhSeH, Et₃N, 38° C., 8 h, 58% (2steps); (iii) 1. HO₂C(CH₂)₅NHBoc (14), EtOCOCl, Et₃N, THF, 0° C., 2.5 h,(acid preactivation), then add to 13, 0° C., 1.5 h; 2. H2 (50 psi), Pd/C(Degussa), THF/EtOH (1:1), 21° C., 24 h; 3. TFA/H2O (4:1), 0° C., 2 h,65% (3 steps); (iv) 4, Et₃N, DMF, 21° C., 2 h, 67%; (v) 5, Et₃N, DMF,21° C., 1.5 h, 75%; (vi) [131I]-NaI, Chloramine-T, MeOH, 21° C., 1 min,55%. Biological evaluation of truncated saponin 16 with three-componentvaccine for (Panel b) anti-KLH (IgG), (Panel c) anti MUC1 (IgG) and(Panel d) anti-OVA (IgG) titers, indicating potent adjuvant activity;horizontal bars indicate median titers; statistical significancecompared to no-adjuvant control: *=p≦0.05, **=p<0.01, ***=p<0.001.(Panel e) Toxicity assessment based on median percent weight loss,indicating low toxicity of 16 (SQS-1-0-5-18); error bars indicatemaximum and minimum values for five mice.

FIG. 4 illustrates oleanolic acid derivative 18, which lacks both theC4-aldehyde substituent and C16-alcohol in the triterpene domain of QS21, exhibits poor adjuvant activity in a preclinical mouse vaccinationmodel. Biological evaluation of oleanolic acid derivative 18(SQS-1-7-5-18) with a three-component vaccine for (Panel a) anti-KLHtiters (IgG), (Panel b) anti MUC1 titers (IgG) and (Panel c) anti-OVAtiters (IgG), indicating attenuated adjuvant activity; horizontal barsindicate median titers; statistical significance compared to no-adjuvantcontrol: *=p≦0.05, **=p<0.01, ***=p<0.001. (Panel d) Toxicity assessmentbased on median percent weight, indicating low toxicity of 18(SQS-1-7-5-18); error bars indicate maximum and minimum values for fivemice.

FIG. 5 illustrates caulophyllogenin derivative 19 and echinocystic acidderivative 20, which lack the C4-aldehyde substituent but retain theC16-alcohol in the triterpene domain of QS-21, exhibit potent adjuvantactivity and no toxicity in a preclinical mouse vaccination model.(Panel a) Structures of saponin adjuvants 19-22 with modifications atthe C4-aldehyde substituent and C16-alcohol of the triterpene domain ofQS 21. The structure in Panel a is shown with6-(4-iodobenzoylamino)-hexaynoyl as the acyl chain. Biologicalevaluation of triterpene variants 19-22 with a four-component vaccine(MUC1-KLH, OVA, GD3 KLH) for (Panel b) anti-KLH (IgG), (Panel c) antiMUC1 (IgG), (Panel d) anti-OVA (IgG), (Panel e) anti-GD3 (IgM), and(Panel f) anti-GD3 (IgG) titers, indicating that the C4-aldehydesubstituent is not required adjuvant activity (19, 20) while removal ofthe C16-alcohol attenuates activity (21, 22); horizontal bars indicatemedian titers; statistical significance compared to no-adjuvant control:*=p≦0.05, **=p<0.01, ***=p<0.001. (Panel g) Toxicity assessment based onmedian percent weight loss, indicating lack of toxicity of 19-22; errorbars indicate maximum and minimum values for five mice.

FIG. 6 illustrates adjuvant-active quillaic acid derivative 16 localizesto the injection site and lymph nodes in mice while adjuvant-attenuatedoleanolic acid derivative 18 does not. In vivo biodistribution in miceof active adjuvant [¹³¹I]⁻¹⁶ ([¹³¹I]-SQS-1-0-5-18) and attenuatedadjuvant [¹³¹I]⁻¹⁸ ([¹³¹I]-SQS-1-7-5-18) at 24 h post-injection in thepresence of 20 μg of OVA; error bars indicate standard deviation frommean for five mice; statistical significance indicated graphically onlyfor lymph nodes and injection site for clarity: *=p≦0.05: lymph nodes,injection site, skin; **=p<0.01: lungs, spleen, stomach, muscle, bone;***=p<0.001: blood, heart.

FIG. 7 illustrates aryl iodide saponin 8 lacking the lineartetrasaccharide domain exhibits poor adjuvant activity in a preclinicalmouse vaccination model. (a) Synthesis of negative control saponin 8(SQS-0-3-7-18): (i) SOCl₂, pyridine, CH2Cl2/DMF, 21° C., 2 h, 91%;(ii) 1. H2 (50 psi), Pd/C (Degussa), THF/EtOH (1:1), 21° C., 24 h; 2.TFA/H2O (4:1), 0° C., 3.3 h, RP-HPLC, 50% (2 steps); (iii) 4, Et₃N, DMF,21° C., 3 h, RP-HPLC, 68%; (iv) 5, Et₃N, DMF, 21° C., 2.5 h, RP-HPLC,53%. (b) Biological evaluation of aryl iodide saponin 8 with OVAantigen. Mice were vaccinated with OVA (20 μg) according to the generalprocedure discussed herein. Median titers represented as red horizontalbars. Statistical significance compared to SQS 21 was assessed usingtwo-tailed unpaired Student's t-test with CI=95%: *=0.01≦p≦0.05(significant).

FIG. 8 illustrates radioiodinated saponin [¹³¹I]-6 localizes to and isretained at the lymph nodes and injection site in mice. Extendedbiodistribution of (a) active radioiodinated saponin [¹³¹I]-6 and (b)inactive radioactive saponin [¹³¹I]-8 with OVA antigen at 24, 72, and 96h post-administration. Significantly higher radioactivity was recoveredin the lymph nodes and at the injection site with [¹³¹I]-6 across allthree timepoints while radioactivity in other organs where a largefold-difference was initially observed (muscle, bone, skin) decreasedrapidly at the later timepoints; the increase in recovery from thethyroid at later timepoints is commonly observed for all radioiodinatedtracers due to deiodination of the tracer. Statistical significance for[¹³¹I]-6 compared to [¹³¹I]-8 in each organ at each timepoint assessedusing two-tailed unpaired Student's t-test with CI=95%. At 24 h:*=0.01≦p≦0.05 (significant): liver, muscle, lymph node, skin, thyroid;**=0.001<p<0.01 (very significant): blood, lungs, spleen, kidneys, bone,injection site; ***=p<0.001 (extremely significant): heart. At 72 h:*=0.01≦p≦0.05 (significant): spleen, thymus, lymph nodes, skin, bone;**=0.001<p<0.01 (very significant): heart, lungs, liver, kidneys,ovaries, thyroid; ***=p<0.001 (extremely significant): blood, injectionsite. At 96 h: *=0.01≦p≦0.05 (significant): bone, ovaries, thymus, skin,thyroid; **=0.001<p<0.01 (very significant): lungs, spleen, stomach,kidney, lymph nodes, injection site; ***=p<0.001 (extremelysignificant): blood, heart, liver.

FIG. 9 illustrates biodistribution of radioiodinated saponins [¹³¹I]-6and [¹³¹I]-8 is not perturbed by the absence of OVA antigen.Biodistribution of (a) active adjuvant [¹³¹I]-6 ([¹³¹I]-SQS-0-0-5-18)and (b) attenuated adjuvant [¹³¹I]-8 ([¹³¹I]-SQS-0-3-7-18). Comparisonof radioactivity recovered in (c) the lymph nodes and (d) at theinjection site, where significantly higher radioactivity was recoveredwith [¹³¹I]-6 across all three timepoints while radioactivity in otherorgans where a large fold-difference was initially observed (muscle,bone, skin) decreased at the later timepoints. Statistical significancefor [¹³¹I]-6 compared to [¹³¹I]-8 in each organ at each timepointassessed using two-tailed unpaired Student's t-test with CI=95%, notshown graphically in parts (a) and (b) for clarity. At 24 h:*=0.01≦p≦0.05 (significant): muscle, bone, ovaries, injection site,skin; **=0.001<p<0.01 (very significant): lungs, liver, spleen, kidneys,thymus; ***=p<0.001 (extremely significant): blood, heart. At 72 h:*=0.01≦p≦0.05 (significant): spleen, thymus, lymph node, injection site;**=0.001<p<0.01 (very significant): blood, lungs, liver, muscle, bone,thyroid; ***=p<0.001 (extremely significant): heart, kidney, ovaries,skin. At 96 h: *=0.01≦p≦0.05 (significant): heart, lungs, liver, spleen,kidney, thymus, lymph node, skin; **=0.001<p<0.01 (very significant):blood, injection site.

FIG. 10 illustrates biodistribution of radioiodinated ovalbumin([¹³¹I]-OVA) indicates rapid deiodination. Biodistribution at 24, 72,and 96 h post-administration in the (a) presence (20 μg) and (b) absenceof active adjuvant 6 (SQS-0-0-5-18). Statistical significance forvaccination with 6 compared to without 6 in each organ at each timepointassessed using two-tailed unpaired Student's t-test with CI=95%, notshown graphically for clarity. At 24 h: *=0.01≦p≦0.05 (significant):heart, skin; **=0.001<p<0.01 (very significant): thymus. At 72 h:*=0.01≦p≦0.05 (significant): stomach, lymph node, skin. At 96 h:*=0.01≦p≦0.05 (significant): blood, lungs, stomach, kidney, bone;**=0.001<p<0.01 (very significant): spleen, ovaries, thymus.

FIG. 11 illustrates fluorescein-labeled active adjuvant 3 is retained atthe injection site. Whole mouse images with fluorescent saponin 3(SQS-0-0-5-12) and amine-containing inactive adjuvant 2 (SQS-0-0-5-11)for comparison to FIG. 2b herein.

FIG. 12 illustrates synthesis of aryl iodide and aryl tin variantsderived from oleanolic acid 18 ([SQS-1-7-5-18]) and S9. (i) 1. TESOTf,2,6-lutidine, CH₂Cl₂, 0° C., 1 h; 2. 12, BF₃.OEt₂, 4 Å M.S., CH₂Cl₂,−50° C., 20 min, 21° C., 2 min [two temperature cycles], 54% (2 steps);(ii) 1. PhSeH, Et₃N, 38° C., 8 h; 2. HO₂C(CH₂)₅NHBoc (14), EtOCOCl,Et₃N, THF, 0° C., 2.5 h, [acid preactivation], then, 0° C., 1.5 h, 77%(2 steps); (iii) 1. H2 (50 psi), Pd/C (Degussa), THF/EtOH (1:1), 21° C.,24 h; 2. TFA/H2O (4:1), 0° C., 2 h, RP-HPLC, 44% (2 steps); (iv) 4,Et₃N, DMF, 21° C., 2 h, RP-HPLC; 63%; (v) 5, Et₃N, DMF, 21° C., 1.5 h,RP-HPLC, 63%; (vi) [131I]-NaI, Chloramine-T, MeOH, 21° C., 1 min,RP-HPLC, 55%.

FIG. 13 illustrates synthesis of aryl iodide saponin adjuvant 19(SQS-1-11-5-18). (i) NaBH₄, MeOH, 21° C., 3 h, >99%; (ii) 1. H2 (1 atm),Pd/C (Degussa), EtOH/THF (1:1), 21° C., 12 h; 2. TFA/H₂O (3:1), 0° C.,1.25 h, RP-HPLC, 70% (2 steps); (iii) 4, Et₃N, DMF, 21° C., 3 h,RP-HPLC, 65%.

FIG. 14 illustrates synthesis of the protected triterpene buildingblocks. (i) TESOTf, 2,6-lutidine, CH₂Cl₂, 0° C., 1 h; S14: 94%; S17:65%; S18: 81%; (ii) 2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPO),N-chlorosuccinimide (NCS), tetrabutylammonium chloride hydrate(TBACl-H₂O), CH₂Cl₂/NaHCO₃ 0.5 M/K₂CO₃ 0.05 M, 21° C., 2 h, 72%.

FIG. 15 illustrates synthesis of additional aryl iodide variants lackingthe branched trisaccharide domain, 20 (SQS-1-8-5-18), 21 (SQS-1-9-5-18),and 22 (SQS-1-10-5-18). (i) S14 or S17 or S18, BF3.OEt2, 4 Å M.S.,CH₂Cl₂, −35° C., 30 min; S19: 80%; S20: 70%; S21: 71%; (ii) 1. PhSeH,Et₃N, 38° C., 8 h; 2. HO₂C(CH₂)₅NHBoc (14), EtOCOCl, Et₃N, THF, 0° C.,2.5 h, [acid preactivation], then, 0° C., 1.5 h; S22: 73% (2 steps);S23: 62% (2 steps); S24: 74%; (iii) 1. H2 (1 atm), Pd/C (Degussa),THF/EtOH (1:1), 21° C., 12 h; 2. TFA/H₂O (3:1), 0° C., 1.25 h, RP-HPLC,S25: 53% (2 steps); S26: 82% (2 steps); S27: 66%; (iv) 4, Et₃N, DMF, 21°C., 3 h, RP-HPLC; 20: 80%; 21: 56%; 22: 57%.

FIG. 16 illustrates complete data for evaluation of triterpene variants19-22 in a preclinical mouse vaccination mode. Biological evaluation of19 (SQS-1-11-5-18), 20 (SQS-1-8-5-18), 21 (SQS-1-9-5-18), and 22(SQS-1-10-5-18) at 20 μg and 50 μg doses with a four-component vaccine(MUC1-KLH, OVA, GD3 KLH) for (a) anti-KLH (IgG), (b) anti MUC1 (IgG),(c) anti-OVA (IgG), (d) anti-GD3 (IgM), and (e) anti-GD3 (IgG) titers.Median titers values represented as red horizontal bars. Statisticalsignificance is compared to no-adjuvant control and was assessed usingtwo-tailed unpaired Student's t test with CI=99%: *=0.01≦p≦0.05(significant), **=0.001<p<0.01 (very significant), ***=p<0.001(extremely significant). (e) Toxicity assessment of 19-22 based onmedian percent weight loss over one week after first vaccine injection.

DETAILED DESCRIPTION

The following detailed description is presented to enable any personskilled in the art to use the present methods and kits. For purposes ofexplanation, specific nomenclature is set forth to provide a thoroughunderstanding of the present methods and kits. However, it will beapparent to one skilled in the art that these specific details are notrequired to practice the use of the methods and kits. Descriptions ofspecific applications are provided only as representative examples. Thepresent methods and kits are not intended to be limited to theembodiments shown, but are to be accorded the widest possible scopeconsistent with the principles and features disclosed herein.

Headings used herein are for organizational purposes only and are notmeant to be used to limit the scope of the description or the claims. Asused throughout this application, the word “may” is used in a permissivesense (i.e., meaning having the potential to), rather than the mandatorysense (i.e., meaning must). The terms “a” and “an” herein do not denotea limitation of quantity, but rather denote the presence of at least oneof the referenced items.

DEFINITIONS

As used herein, the following definitions shall apply unless otherwiseindicated.

The term “aliphatic” or “aliphatic group,” as used herein, means astraight-chain (i.e., unbranched) or branched, substituted orunsubstituted hydrocarbon chain that is completely saturated or thatcontains one or more units of unsaturation, or a monocyclic hydrocarbonor bicyclic hydrocarbon that is completely saturated or that containsone or more units of unsaturation, but which is not aromatic (alsoreferred to herein as “carbocycle,” “cycloaliphatic” or “cycloalkyl”),that has a single point of attachment to the rest of the molecule.Unless otherwise specified, aliphatic groups contain 1-12 aliphaticcarbon atoms. In some embodiments, aliphatic groups contain 1-6aliphatic carbon atoms. In some embodiments, aliphatic groups contain1-5 aliphatic carbon atoms. In other embodiments, aliphatic groupscontain 1-4 aliphatic carbon atoms. In still other embodiments,aliphatic groups contain 1-3 aliphatic carbon atoms, and in yet otherembodiments, aliphatic groups contain 1-2 aliphatic carbon atoms. Insome embodiments, “cycloaliphatic” (or “carbocycle” or “cycloalkyl”)refers to a monocyclic C₃-C₆ hydrocarbon that is completely saturated orthat contains one or more units of unsaturation, but which is notaromatic, that has a single point of attachment to the rest of themolecule. Suitable aliphatic groups include, but are not limited to,linear or branched, substituted or unsubstituted alkyl, alkenyl, alkynylgroups and hybrids thereof such as (cycloalkyl)alkyl,(cycloalkenyl)alkyl or (cycloalkyl)alkenyl.

The term “lower alkyl” refers to a C₁₋₄ straight or branched alkylgroup. Exemplary lower alkyl groups are methyl, ethyl, propyl,isopropyl, butyl, isobutyl, and tert-butyl.

The term “lower haloalkyl” refers to a C₁₋₄ straight or branched alkylgroup that is substituted with one or more halogen atoms.

The term “heteroatom” means one or more of oxygen, sulfur, nitrogen,phosphorus, or silicon (including, any oxidized form of nitrogen,sulfur, phosphorus, or silicon; the quaternized form of any basicnitrogen or; a substitutable nitrogen of a heterocyclic ring, forexample N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl) orNR⁺ (as in N-substituted pyrrolidinyl)).

The term “unsaturated,” as used herein, means that a moiety has one ormore units of unsaturation.

As used herein, the term “bivalent C₁₋₁₂ (or C₁₋₂₆, C₁₋₁₆, C₁₋₈) orsaturated or unsaturated, straight or branched, hydrocarbon chain,”refers to bivalent alkylene, alkenylene, and alkynylene chains that arestraight or branched as defined herein.

The term “alkylene” refers to a bivalent alkyl group. An “alkylenechain” is a polymethylene group, i.e., —(CH₂)_(n)—, wherein n is apositive integer, preferably from 1 to 6, from 1 to 4, from 1 to 3, from1 to 2, or from 2 to 3. A substituted alkylene chain is a polymethylenegroup in which one or more methylene hydrogen atoms are replaced with asubstituent. Suitable substituents include those described below for asubstituted aliphatic group.

The term “alkenylene” refers to a bivalent alkenyl group. A substitutedalkenylene chain is a polymethylene group containing at least one doublebond in which one or more hydrogen atoms are replaced with asubstituent. Suitable substituents include those described below for asubstituted aliphatic group.

The term “alkynylene” refers to a bivalent alkynyl group. A substitutedalkynylene chain is a polymethylene group containing at least one doublebond in which one or more hydrogen atoms are replaced with asubstituent. Suitable substituents include those described below for asubstituted aliphatic group.

The term “acyl,” used alone or a part of a larger moiety, refers togroups formed by removing a hydroxy group from a carboxylic acid.

The term “halogen” means F, Cl, Br, or I.

The terms “aralkyl” and “arylalkyl” are used interchangably and refer toalkyl groups in which a hydrogen atom has been replaced with an arylgroup. Such groups include, without limitation, benzyl, cinnamyl, anddihyrocinnamyl.

The term “aryl” used alone or as part of a larger moiety as in“aralkyl,” “aralkoxy,” or “aryloxyalkyl,” refers to monocyclic orbicyclic ring systems having a total of five to fourteen ring members,wherein at least one ring in the system is aromatic and wherein eachring in the system contains 3 to 7 ring members. The term “aryl” may beused interchangeably with the term “aryl ring.”

In certain embodiments of the present invention, “aryl” refers to anaromatic ring system which includes, but not limited to, phenyl,biphenyl, naphthyl, anthracyl and the like, which may bear one or moresubstituents. Also included within the scope of the term “aryl,” as itis used herein, is a group in which an aromatic ring is fused to one ormore non-aromatic rings, such as indanyl, phthalimidyl, naphthimidyl,phenanthridinyl, or tetrahydronaphthyl, and the like.

The terms “heteroaryl” and “heteroar-,” used alone or as part of alarger moiety, e.g., “heteroaralkyl,” or “heteroaralkoxy,” refer togroups having 5 to 10 ring atoms, preferably 5, 6, or 9 ring atoms;having 6, 10, or 14 π electrons shared in a cyclic array; and having, inaddition to carbon atoms, from one to five heteroatoms. The term“heteroatom” refers to nitrogen, oxygen, or sulfur, and includes anyoxidized form of nitrogen or sulfur, and any quaternized form of a basicnitrogen. Heteroaryl groups include, without limitation, thienyl,furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl,oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl,thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl,purinyl, naphthyridinyl, and pteridinyl. The terms “heteroaryl” and“heteroar-”, as used herein, also include groups in which aheteroaromatic ring is fused to one or more aryl, cycloaliphatic, orheterocyclyl rings, where the radical or point of attachment is on theheteroaromatic ring. Nonlimiting examples include indolyl, isoindolyl,benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl,benzthiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl,quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl,phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl,tetrahydroisoquinolinyl, and pyrido[2,3-b]-1,4-oxazin-3(4H)-one. Aheteroaryl group may be mono- or bicyclic. The term “heteroaryl” may beused interchangeably with the terms “heteroaryl ring,” “heteroarylgroup,” or “heteroaromatic,” any of which terms include rings that areoptionally substituted. The terms “heteroaralkyl” and “heteroarylalkyl”refer to an alkyl group substituted by a heteroaryl moiety, wherein thealkyl and heteroaryl portions independently are optionally substituted.

The term “heteroaliphatic,” as used herein, means aliphatic groupswherein one or two carbon atoms are independently replaced by one ormore of oxygen, sulfur, nitrogen, or phosphorus. Heteroaliphatic groupsmay be substituted or unsubstituted, branched or unbranched, cyclic oracyclic, and include “heterocycle,” “hetercyclyl,”“heterocycloaliphatic,” or “heterocyclic” groups.

As used herein, the terms “heterocycle,” “heterocyclyl,” “heterocyclicradical,” and “heterocyclic ring” are used interchangeably and refer toa stable 5- to 7-membered monocyclic or 7-10-membered bicyclicheterocyclic moiety that is either saturated or partially unsaturated,and having, in addition to carbon atoms, one or more, preferably one tofour, heteroatoms, as defined above. When used in reference to a ringatom of a heterocycle, the term “nitrogen” includes a substitutednitrogen. As an example, in a saturated or partially unsaturated ringhaving 0-3 heteroatoms selected from oxygen, sulfur or nitrogen, thenitrogen may be N (as in 3,4-dihydro-2H-pyrrolyl), NH (as inpyrrolidinyl), or ⁺NR (as in N-substituted pyrrolidinyl).

A heterocyclic ring can be attached to its pendant group at anyheteroatom or carbon atom that results in a stable structure and any ofthe ring atoms can be optionally substituted. Examples of such saturatedor partially unsaturated heterocyclic radicals include, withoutlimitation, tetrahydrofuranyl, tetrahydrothiophenyl pyrrolidinyl,piperidinyl, pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl,decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl,diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and quinuclidinyl. Theterms “heterocycle,” “heterocyclyl,” “heterocyclyl ring,” “heterocyclicgroup,” “heterocyclic moiety,” and “heterocyclic radical,” are usedinterchangeably herein, and also include groups in which a heterocyclylring is fused to one or more aryl, heteroaryl, or cycloaliphatic rings,such as indolinyl, 3H-indolyl, chromanyl, phenanthridinyl, ortetrahydroquinolinyl, where the radical or point of attachment is on theheterocyclyl ring. A heterocyclyl group may be mono- or bicyclic. Theterm “heterocyclylalkyl” refers to an alkyl group substituted by aheterocyclyl, wherein the alkyl and heterocyclyl portions independentlyare optionally substituted.

As used herein, the term “partially unsaturated” refers to a ring moietythat includes at least one double or triple bond. The term “partiallyunsaturated” is intended to encompass rings having multiple sites ofunsaturation, but is not intended to include aryl or heteroarylmoieties, as herein defined.

The phrase “pharmaceutically acceptable carrier” as used herein means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, or solvent encapsulatingmaterial, involved in carrying or transporting the subject compound fromone organ, or portion of the body, to another organ, or portion of thebody. Each carrier must be “acceptable” in the sense of being compatiblewith the other ingredients of the formulation and not injurious to thepatient. Some examples of materials which can serve aspharmaceutically-acceptable carriers include: sugars, such as lactose,glucose and sucrose; starches, such as corn starch and potato starch;cellulose, and its derivatives, such as sodium carboxymethyl cellulose,ethyl cellulose and cellulose acetate; powdered tragacanth; malt;gelatin; talc; excipients, such as cocoa butter and suppository waxes;oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil,olive oil, corn oil and soybean oil; glycols, such as propylene glycol;polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol;esters, such as ethyl oleate and ethyl laurate; agar; buffering agents,such as magnesium hydroxide and aluminum hydroxide; alginic acid;pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol;pH buffered solutions; polyesters, polycarbonates and/or polyanhydrides;and other non-toxic compatible substances employed in pharmaceuticalformulations.

As used herein, the term “pharmaceutically acceptable salt” refers tothose salts which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of humans and lower animalswithout undue toxicity, irritation, allergic response and the like, andare commensurate with a reasonable benefit/risk ratio. Pharmaceuticallyacceptable salts are well known in the art. For example, S. M. Berge etal., describe pharmaceutically acceptable salts in detail in J.Pharmaceutical Sciences, 1977, 66, 1-19, incorporated herein byreference. Pharmaceutically acceptable salts of the compounds of thisinvention include those derived from suitable inorganic and organicacids and bases. Examples of pharmaceutically acceptable, nontoxic acidaddition salts are salts of an amino group formed with inorganic acidssuch as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuricacid and perchloric acid or with organic acids such as acetic acid,oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid ormalonic acid or by using other methods used in the art such as ionexchange. Other pharmaceutically acceptable salts include adipate,alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate,borate, butyrate, camphorate, camphorsulfonate, citrate,cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,formate, fumarate, glucoheptonate, glycerophosphate, gluconate,hemisulfate, heptanoate, hexanoate, hydroiodide,2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, laurylsulfate, malate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, pivalate,propionate, stearate, succinate, sulfate, tartrate, thiocyanate,p-toluenesulfonate, undecanoate, valerate salts, and the like.

In other cases, the compounds of the present invention may contain oneor more acidic functional groups and, thus, are capable of formingpharmaceutically-acceptable salts with pharmaceutically acceptablebases. The term “pharmaceutically acceptable salts” in these instancesrefers to the relatively non-toxic, inorganic and organic base additionsalts of compounds of the present invention. These salts can likewise beprepared in situ in the administration vehicle or the dosage formmanufacturing process, or by separately reacting the purified compoundin its free acid form with a suitable base, such as the hydroxide,carbonate or bicarbonate of a pharmaceutically acceptable metal cation,with ammonia, or with a pharmaceutically acceptable organic primary,secondary, tertiary, or quaternary amine. Salts derived from appropriatebases include alkali metal, alkaline earth metal, ammonium andN⁺(C₁₋₄alkyl)₄ salts. Representative alkali or alkaline earth metalsalts include sodium, lithium, potassium, calcium, magnesium, and thelike. Further pharmaceutically acceptable salts include, whenappropriate, nontoxic ammonium, quaternary ammonium, and amine cationsformed using counterions such as halide, hydroxide, carboxylate,sulfate, phosphate, nitrate, loweralkyl sulfonate and aryl sulfonate.Representative organic amines useful for the formation of base additionsalts include ethylamine, diethylamine, ethylenediamine, ethanolamine,diethanolamine, piperazine and the like. (See, for example, Berge etal., supra).

Unless otherwise stated, structures depicted herein are also meant toinclude all isomeric (e.g., enantiomeric, diastereomeric, and geometric(or conformational)) forms of the structure; for example, the R and Sconfigurations for each stereocenter, Z and E double bond isomers, and Zand E conformational isomers. Therefore, single stereochemical isomersas well as enantiomeric, diastereomeric, and geometric (orconformational) mixtures of the present compounds are within the scopeof the invention. Unless otherwise stated, all tautomeric forms of thecompounds of the invention are within the scope of the invention.

Provided compounds may comprise one or more saccharide moieties. Unlessotherwise specified, both D- and L-configurations, and mixtures thereof,are within the scope of the invention. Unless otherwise specified, bothα- and β-linked embodiments, and mixtures thereof, are contemplated bythe present invention.

If, for instance, a particular enantiomer of a compound of the presentinvention is desired, it may be prepared by asymmetric synthesis, chiralchromatography, or by derivation with a chiral auxiliary, where theresulting diastereomeric mixture is separated and the auxiliary groupcleaved to provide the pure desired enantiomers. Alternatively, wherethe molecule contains a basic functional group, such as amino, or anacidic functional group, such as carboxyl, diastereomeric salts areformed with an appropriate optically-active acid or base, followed byresolution of the diastereomers thus formed by fractionalcrystallization or chromatographic means well known in the art, andsubsequent recovery of the pure enantiomers.

Additionally, unless otherwise stated, structures depicted herein arealso meant to include compounds that differ only in the presence of oneor more isotopically enriched atoms. For example, compounds having thepresent structures including the replacement of hydrogen by deuterium ortritium, or the replacement of a carbon by a ¹³C- or ¹⁴C-enriched carbonare within the scope of this invention. Such compounds are useful, forexample, as analytical tools, as probes in biological assays, or astherapeutic agents in accordance with the present invention.

As used herein, the term “reducing agent” refers to a reagent suitablefor carrying out the contextually relevant reduction reaction.Exemplary, but non-limiting, reducing agents are: lithium aluminumhydride (LiAlH4), sodium borohydride (NaBH4), hydroboration reagents(BH3, B2H6), alkali metals (e.g. Li or Na), transition metals (e.g. Sn,Zn, or Fe), Grignard reagents (RMgX), and organometallics (Rli, RNa,R2CuLi). The term may also encompass reductive techniques such ascatalytic hydrogenation. One of ordinary skill in the art willappreciate that the synthetic methods, as described herein, utilize avariety of reducing agents.

One of ordinary skill in the art will appreciate that the syntheticmethods, as described herein, utilize a variety of protecting groups. Bythe term “protecting group,” as used herein, it is meant that aparticular functional moiety, e.g., O, S, or N, is masked or blocked,permitting, if desired, a reaction to be carried out selectively atanother reactive site in a multifunctional compound. In preferredembodiments, a protecting group reacts selectively in good yield to givea protected substrate that is stable to the projected reactions; theprotecting group is preferably selectively removable by readilyavailable, preferably non-toxic reagents that do not attack the otherfunctional groups; the protecting group forms a separable derivative(more preferably without the generation of new stereogenic centers); andthe protecting group will preferably have a minimum of additionalfunctionality to avoid further sites of reaction. As detailed herein,oxygen, sulfur, nitrogen, and carbon protecting groups may be utilized.By way of non-limiting example, hydroxyl protecting groups includemethyl, methoxylmethyl (MOM), methylthiomethyl (MTM), t-butylthiomethyl,(phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM),p-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM),guaiacolmethyl (GUM), t-butoxymethyl, 4-pentenyloxymethyl (POM),siloxymethyl, 2-methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl,bis(2-chloroethoxy)methyl, 2-(trimethylsilyl)ethoxymethyl (SEMOR),tetrahydropyranyl (THP), 3-bromotetrahydropyranyl,tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4-methoxytetrahydropyranyl(MTHP), 4-methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranylS,S-dioxide, 1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl(CTMP), 1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl,2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl,1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl,1-methyl-1-benzyloxyethyl, 1-methyl-1-benzyloxy-2-fluoroethyl,2,2,2-trichloroethyl, 2-trimethylsilylethyl, 2-(phenylselenyl)ethyl,t-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl,benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl,p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl,p-phenylbenzyl, 2-picolyl, 4-picolyl, 3-methyl-2-picolyl N-oxido,diphenylmethyl, p,p′-dinitrobenzhydryl, 5-dibenzosuberyl,triphenylmethyl, α-naphthyldiphenylmethyl,p-methoxyphenyldiphenylmethyl, di(p-methoxyphenyl)phenylmethyl,tri(p-methoxyphenyl)methyl, 4-(4′-bromophenacyloxyphenyl)diphenylmethyl,4,4′,4″-tris(4,5-dichlorophthalimidophenyl)methyl,4,4′,4″-tris(levulinoyloxyphenyl)methyl,4,4′,4″-tris(benzoyloxyphenyl)methyl,3-(imidazol-1-yl)bis(4′,4″-dimethoxyphenyl)methyl,1,1-bis(4-methoxyphenyl)-1′-pyrenylmethyl, 9-anthryl,9-(9-phenyl)xanthenyl, 9-(9-phenyl-10-oxo)anthryl,1,3-benzodithiolan-2-yl, benzisothiazolyl S,S-dioxido, trimethylsilyl(TMS), triethylsilyl (TES), triisopropylsilyl (TIPS),dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEEPS),dimethylthexylsilyl, t-butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl(TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl,diphenylmethylsilyl (DPMS), t-butylmethoxyphenylsilyl (TBMPS), formate,benzoylformate, acetate, chloroacetate, dichloroacetate,trichloroacetate, trifluoroacetate, methoxyacetate,triphenylmethoxyacetate, phenoxyacetate, p-chlorophenoxyacetate,3-phenylpropionate, 4-oxopentanoate (levulinate),4,4-(ethylenedithio)pentanoate (levulinoyldithioacetal), pivaloate,adamantoate, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate,2,4,6-trimethylbenzoate (mesitoate), alkyl methyl carbonate,9-fluorenylmethyl carbonate (Fmoc), alkyl ethyl carbonate, alkyl2,2,2-trichloroethyl carbonate (Troc), 2-(trimethylsilyl)ethyl carbonate(TMSEC), 2-(phenylsulfonyl) ethyl carbonate (Psec),2-(triphenylphosphonio) ethyl carbonate (Peoc), alkyl isobutylcarbonate, alkyl vinyl carbonate alkyl allyl carbonate, alkylp-nitrophenyl carbonate, alkyl benzyl carbonate, alkyl p-methoxybenzylcarbonate, alkyl 3,4-dimethoxybenzyl carbonate, alkyl o-nitrobenzylcarbonate, alkyl p-nitrobenzyl carbonate, alkyl S-benzyl thiocarbonate,4-ethoxy-1-napththyl carbonate, methyl dithiocarbonate, 2-iodobenzoate,4-azidobutyrate, 4-nitro-4-methylpentanoate, o-(dibromomethyl)benzoate,2-formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl,4-(methylthiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoate,2,6-dichloro-4-methylphenoxyacetate,2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetate,2,4-bis(1,1-dimethylpropyl)phenoxyacetate, chlorodiphenylacetate,isobutyrate, monosuccinoate, (E)-2-methyl-2-butenoate,o-(methoxycarbonyl)benzoate, α-naphthoate, nitrate, alkylN,N,N′,N′-tetramethylphosphorodiamidate, alkyl N-phenylcarbamate,borate, dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate,sulfate, methanesulfonate (mesylate), benzylsulfonate, and tosylate(Ts). For protecting 1,2- or 1,3-diols, the protecting groups includemethylene acetal, ethylidene acetal, 1-t-butylethylidene ketal,1-phenylethylidene ketal, (4-methoxyphenyl)ethylidene acetal,2,2,2-trichloroethylidene acetal, acetonide, cyclopentylidene ketal,cyclohexylidene ketal, cycloheptylidene ketal, benzylidene acetal,p-methoxybenzylidene acetal, 2,4-dimethoxybenzylidene ketal,3,4-dimethoxybenzylidene acetal, 2-nitrobenzylidene acetal,methoxymethylene acetal, ethoxymethylene acetal, dimethoxymethyleneortho ester, 1-methoxyethylidene ortho ester, 1-ethoxyethylidine orthoester, 1,2-dimethoxyethylidene ortho ester, α-methoxybenzylidene orthoester, 1-(N,N-dimethylamino)ethylidene derivative,α-(N,N′-dimethylamino)benzylidene derivative, 2-oxacyclopentylideneortho ester, di-t-butylsilylene group (DTBS),tetraisopropyldisiloxanylidene) derivative (TIPDS),tetra-t-butoxydisiloxane-1,3-diylidene derivative (TBDS), cycliccarbonates, cyclic boronates, ethyl boronate, and phenyl boronate.Amino-protecting groups include methyl carbamate, ethyl carbamante,9-fluorenylmethyl carbamate (Fmoc), 9-(2-sulfo)fluorenylmethylcarbamate, 9-(2,7-dibromo)fluoroenylmethyl carbamate,2,7-di-t-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methylcarbamate (DBD-Tmoc), 4-methoxyphenacyl carbamate (Phenoc),2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl carbamate(Teoc), 2-phenylethyl carbamate (hZ), 1-(1-adamantyl)-1-methylethylcarbamate (Adpoc), 1,1-dimethyl-2-haloethyl carbamate,1,1-dimethyl-2,2-dibromoethyl carbamate (DB-t-BOC),1,1-dimethyl-2,2,2-trichloroethyl carbamate (TCBOC),1-methyl-1-(4-biphenylyl)ethyl carbamate (Bpoc),1-(3,5-di-t-butylphenyl)-1-methylethyl carbamate (t-Bumeoc), 2-(2′- and4′-pyridyl)ethyl carbamate (Pyoc), 2-(N,N-dicyclohexylcarboxamido)ethylcarbamate, t-butyl carbamate (BOC), 1-adamantyl carbamate (Adoc), vinylcarbamate (Voc), allyl carbamate (Alloc), 1-isopropylallyl carbamate(Ipaoc), cinnamyl carbamate (Coc), 4-nitrocinnamyl carbamate (Noc),8-quinolyl carbamate, N-hydroxypiperidinyl carbamate, alkyldithiocarbamate, benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz),p-nitobenzyl carbamate, p-bromobenzyl carbamate, p-chlorobenzylcarbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfinylbenzylcarbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl carbamate,2-methylthioethyl carbamate, 2-methylsulfonylethyl carbamate,2-(p-toluenesulfonyl)ethyl carbamate, [2-(1,3-dithianyl)]methylcarbamate (Dmoc), 4-methylthiophenyl carbamate (Mtpc),2,4-dimethylthiophenyl carbamate (Bmpc), 2-phosphonioethyl carbamate(Peoc), 2-triphenylphosphonioisopropyl carbamate (Ppoc),1,1-dimethyl-2-cyanoethyl carbamate, m-chloro-p-acyloxybenzyl carbamate,p-(dihydroxyboryl)benzyl carbamate, 5-benzisoxazolylmethyl carbamate,2-(trifluoromethyl)-6-chromonylmethyl carbamate (Tcroc), m-nitrophenylcarbamate, 3,5-dimethoxybenzyl carbamate, o-nitrobenzyl carbamate,3,4-dimethoxy-6-nitrobenzyl carbamate, phenyl(o-nitrophenyl)methylcarbamate, phenothiazinyl-(10)-carbonyl derivative,N′-p-toluenesulfonylaminocarbonyl derivative, N′-phenylaminothiocarbonylderivative, t-amyl carbamate, S-benzyl thiocarbamate, p-cyanobenzylcarbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentylcarbamate, cyclopropylmethyl carbamate, p-decyloxybenzyl carbamate,2,2-dimethoxycarbonylvinyl carbamate, o-(N,N-dimethylcarboxamido)benzylcarbamate, 1,1-dimethyl-3-(N,N-dimethylcarboxamido)propyl carbamate,1,1-dimethylpropynyl carbamate, di(2-pyridyl)methyl carbamate,2-furanylmethyl carbamate, 2-iodoethyl carbamate, isoborynl carbamate,isobutyl carbamate, isonicotinyl carbamate,p-(p′-methoxyphenylazo)benzyl carbamate, 1-methylcyclobutyl carbamate,1-methylcyclohexyl carbamate, 1-methyl-1-cyclopropylmethyl carbamate,1-methyl-1-(3,5-dimethoxyphenyl)ethyl carbamate,1-methyl-1-(p-phenylazophenyl)ethyl carbamate, 1-methyl-1-phenylethylcarbamate, 1-methyl-1-(4-pyridyl)ethyl carbamate, phenyl carbamate,p-(phenylazo)benzyl carbamate, 2,4,6-tri-t-butylphenyl carbamate,4-(trimethylammonium)benzyl carbamate, 2,4,6-trimethylbenzyl carbamate,formamide, acetamide, chloroacetamide, trichloroacetamide,trifluoroacetamide, phenylacetamide, 3-phenylpropanamide, picolinamide,3-pyridylcarboxamide, N-benzoylphenylalanyl derivative, benzamide,p-phenylbenzamide, o-nitophenylacetamide, o-nitrophenoxyacetamide,acetoacetamide, (N′-dithiobenzyloxycarbonylamino)acetamide,3-(p-hydroxyphenyl)propanamide, 3-(o-nitrophenyl)propanamide,2-methyl-2-(o-nitrophenoxy)propanamide,2-methyl-2-(o-phenylazophenoxy)propanamide, 4-chlorobutanamide,3-methyl-3-nitrobutanamide, o-nitrocinnamide, N-acetylmethioninederivative, o-nitrobenzamide, o-(benzoyloxymethyl)benzamide,4,5-diphenyl-3-oxazolin-2-one, N-phthalimide, N-dithiasuccinimide (Dts),N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole,N-1,1,4,4-tetramethyldisilylazacyclopentane adduct (STABASE),5-substituted 1,3-dimethyl-1,3,5-triazacyclohexan-2-one, 5-substituted1,3-dibenzyl-1,3,5-triazacyclohexan-2-one, 1-substituted3,5-dinitro-4-pyridone, N-methylamine, N-allylamine,N-[2-(trimethylsilyl)ethoxy]methylamine (SEM), N-3-acetoxypropylamine,N-(1-isopropyl-4-nitro-2-oxo-3-pyroolin-3-yl)amine, quaternary ammoniumsalts, N-benzylamine, N-di(4-methoxyphenyl)methylamine,N-5-dibenzosuberylamine, N-triphenylmethylamine (Tr),N-[(4-methoxyphenyl)diphenylmethyl]amine (MMTr),N-9-phenylfluorenylamine (PhF),N-2,7-dichloro-9-fluorenylmethyleneamine, N-ferrocenylmethylamino (Fcm),N-2-picolylamino N′-oxide, N-1,1-dimethylthiomethyleneamine,N-benzylideneamine, N-p-methoxybenzylideneamine,N-diphenylmethyleneamine, N-[(2-pyridyl)mesityl]methyleneamine,N-(N′,N′-dimethylaminomethylene)amine, N,N′-isopropylidenediamine,N-p-nitrobenzylideneamine, N-salicylideneamine,N-5-chlorosalicylideneamine,N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine,N-cyclohexylideneamine, N-(5,5-dimethyl-3-oxo-1-cyclohexenyl)amine,N-borane derivative, N-diphenylborinic acid derivative,N-[phenyl(pentacarbonylchromium- or tungsten)carbonyl]amine, N-copperchelate, N-zinc chelate, N-nitroamine, N-nitrosoamine, amine N-oxide,diphenylphosphinamide (Dpp), dimethylthiophosphinamide (Mpt),diphenylthiophosphinamide (Ppt), dialkyl phosphoramidates, dibenzylphosphoramidate, diphenyl phosphoramidate, benzenesulfenamide,o-nitrobenzenesulfenamide (Nps), 2,4-dinitrobenzenesulfenamide,pentachlorobenzenesulfenamide, 2-nitro-4-methoxybenzenesulfenamide,triphenylmethylsulfenamide, 3-nitropyridinesulfenamide (Npys),p-toluenesulfonamide (Ts), benzenesulfonamide,2,3,6,-trimethyl-4-methoxybenzenesulfonamide (Mtr),2,4,6-trimethoxybenzenesulfonamide (Mtb),2,6-dimethyl-4-methoxybenzenesulfonamide (Pme),2,3,5,6-tetramethyl-4-methoxybenzenesulfonamide (Mte),4-methoxybenzenesulfonamide (Mbs), 2,4,6-trimethylbenzenesulfonamide(Mts), 2,6-dimethoxy-4-methylbenzenesulfonamide (iMds),2,2,5,7,8-pentamethylchroman-6-sulfonamide (Pmc), methanesulfonamide(Ms), β-trimethylsilylethanesulfonamide (SES), 9-anthracenesulfonamide,4-(4′,8′-dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS),benzylsulfonamide, trifluoromethylsulfonamide, and phenacylsulfonamide.Exemplary protecting groups are detailed herein, however, it will beappreciated that the present invention is not intended to be limited tothese protecting groups; rather, a variety of additional equivalentprotecting groups can be readily identified using the above criteria andutilized in the method of the present invention. Additionally, a varietyof protecting groups are described by Greene and Wuts (supra).

As described herein, compounds of the invention may contain “optionallysubstituted” moieties. In general, the term “substituted,” whetherpreceded by the term “optionally” or not, means that one or morehydrogens of the designated moiety are replaced with a suitablesubstituent. Unless otherwise indicated, an “optionally substituted”group may have a suitable substituent at each substitutable position ofthe group, and when more than one position in any given structure may besubstituted with more than one substituent selected from a specifiedgroup, the substituent may be either the same or different at everyposition. Combinations of substituents envisioned by this invention arepreferably those that result in the formation of stable or chemicallyfeasible compounds. The term “stable,” as used herein, refers tocompounds that are not substantially altered when subjected toconditions to allow for their production, detection, and, in certainembodiments, their recovery, purification, and use for one or more ofthe purposes disclosed herein.

Suitable monovalent substituents on a substitutable carbon atom of an“optionally substituted” group are independently halogen;—(CH₂)₀₋₄R^(∘); —(CH₂)₀₋₄OR^(∘); —O(CH₂)₀₋₄R^(∘), —O—(CH₂)₀₋₄C(O)OR^(∘);—(CH₂)₀₋₄CH(OR^(∘))₂; —(CH₂)₀₋₄SR^(∘); —(CH₂)₀₋₄Ph, which may besubstituted with R^(∘); —(CH₂)₀₋₄O(CH₂)₀₋₁Ph which may be substitutedwith R^(∘); —CH═CHPh, which may be substituted with R^(∘);—(CH₂)₀₋₄O(CH₂)₀₋₁-pyridyl which may be substituted with R^(∘); —NO₂;—CN; —N₃; —(CH₂)₀₋₄N(R^(∘))₂; —(CH₂)₀₋₄N(R^(∘))C(O)R^(∘);—N(R^(∘))C(S)R^(∘); —(CH₂)₀₋₄N(R^(∘))C(O)NR^(∘) ₂; —N(R^(∘))C(S)NR^(∘)₂; —(CH₂)₀₋₄N(R^(∘))C(O)OR^(∘); —N(R^(∘))N(R^(∘))C(O)R^(∘);—N(R^(∘))N(R^(∘))C(O)NR^(∘) ₂; —N(R^(∘))N(R^(∘))C(O)OR^(∘);—(CH₂)₀₋₄C(O)R^(∘); —C(S)R^(∘); —(CH₂)₀₋₄C(O)OR^(∘);—(CH₂)₀₋₄C(O)SR^(∘); —(CH₂)₀₋₄C(O)OSiR^(∘) ₃; —(CH₂)₀₋₄OC(O)R^(∘);—OC(O)(CH₂)₀₋₄SR—, SC(S)SR^(∘); —(CH₂)₀₋₄SC(O)R^(∘); —(CH₂)₀₋₄C(O)NR^(∘)₂; —C(S)NR^(∘) ₂; —C(S)SR^(∘); —SC(S)SR^(∘), —(CH₂)₀₋₄OC(O)NR^(∘) ₂;—C(O)N(OR^(∘))R^(∘); —C(O)C(O)R^(∘); —C(O)CH₂C(O)R^(∘);—C(NOR^(∘))R^(∘); —(CH₂)₀₋₄SSR^(∘); —(CH₂)₀₋₄S(O)₂R^(∘);—(CH₂)₀₋₄S(O)₂OR^(∘); —(CH₂)₀₋₄OS(O)₂R^(∘); —S(O)₂NR^(∘) ₂;—(CH₂)₀₋₄S(O)R^(∘); —N(R^(∘))S(O)₂NR^(∘) ₂; —N(R^(∘))S(O)₂R^(∘);—N(OR^(∘))R^(∘); —C(NH)NR^(∘) ₂; —P(O)₂R^(∘); —P(O)R^(∘) ₂; —OP(O)R^(∘)₂; —OP(O)(OR^(∘))₂; SiR^(∘) ₃; —(C₁₋₄ straight orbranched)alkylene)O—N(R^(∘) ₂; or —(C₁₋₄ straight or branchedalkylene)C(O)O—N(R^(∘))₂, wherein each R^(∘) may be substituted asdefined below and is independently hydrogen, C₁₋₆ aliphatic, —CH₂Ph,—O(CH₂)₀₋₁Ph, —CH₂-(5-6-membered heteroaryl ring), or a 5-6-memberedsaturated, partially unsaturated, or aryl ring having 0-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, or,notwithstanding the definition above, two independent occurrences ofR^(∘), taken together with their intervening atom(s), form a3-12-membered saturated, partially unsaturated, or aryl mono- orbicyclic ring having 0-4 heteroatoms independently selected fromnitrogen, oxygen, or sulfur, which may be substituted as defined below.

Suitable monovalent substituents on R^(∘) (or the ring formed by takingtwo independent occurrences of R^(∘) together with their interveningatoms), are independently halogen, —(CH₂)₀₋₂R^(•), -(haloR^(•)),—(CH₂)₀₋₂OH, —(CH₂)₀₋₂OR^(•), —(CH₂)₀₋₂CH(OR^(•))₂; —O(haloR^(•)), —CN,—N₃, —(CH₂)₀₋₂C(O)R^(•), —(CH₂)₀₋₂C(O)OH, —(CH₂)₀₋₂C(O)OR^(•),—(CH₂)₀₋₂SR^(•), —(CH₂)₀₋₂SH, —(CH₂)₀₋₂NH₂, —(CH₂)₀₋₂NHR^(•),—(CH₂)₀₋₂NR^(•) ₂, —NO₂, —SiR^(•) ₃, —OSiR^(•) ₃, —C(O)SR^(•), —(C₁₋₄straight or branched alkylene)C(O)OR^(•), or —SSR^(•) wherein each R^(•)is unsubstituted or where preceded by “halo” is substituted only withone or more halogens, and is independently selected from C₁₋₄ aliphatic,—CH₂Ph, —O(CH₂)₀₋₁Ph, or a 5-6-membered saturated, partiallyunsaturated, or aryl ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur. Suitable divalent substituents on asaturated carbon atom of R^(∘) include ═O and ═S.

Suitable divalent substituents on a saturated carbon atom of an“optionally substituted” group include the following: ═O, ═S, ═NNR*₂,═NNHC(O)R*, ═NNHC(O)OR*, ═NNHS(O)₂R*, ═NR*, ═NOR*, —O(C(R*₂))₂₋₃O—, or—S(C(R*₂))₂₋₃S—, wherein each independent occurrence of R* is selectedfrom hydrogen, C₁₋₆ aliphatic which may be substituted as defined below,or an unsubstituted 5-6-membered saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur. Suitable divalent substituents that are bound tovicinal substitutable carbons of an “optionally substituted” groupinclude: —O(CR*₂)₂₋₃O—, wherein each independent occurrence of R* isselected from hydrogen, C₁₋₆ aliphatic which may be substituted asdefined below, or an unsubstituted 5-6-membered saturated, partiallyunsaturated, or aryl ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur.

Suitable substituents on the aliphatic group of R* include halogen,—R^(•), -(haloR^(•)), —OH, —OR^(•), —O(haloR^(•)), —CN, —C(O)OH,—C(O)OR^(•), —NH₂, —NHR^(•), —NR^(•) ₂, or —NO₂, wherein each R^(•) isunsubstituted or where preceded by “halo” is substituted only with oneor more halogens, and is independently C₁₋₄ aliphatic, —CH₂Ph,—O(CH₂)₀₋₁Ph, or a 5-6-membered saturated, partially unsaturated, oraryl ring having 0-4 heteroatoms independently selected from nitrogen,oxygen, or sulfur.

Suitable substituents on a substitutable nitrogen of an “optionallysubstituted” group include —R^(†), —NR^(†) ₂, —C(O)R^(†), —C(O)OR^(†),—C(O)C(O)R^(†), —C(O)CH₂C(O)R^(†), —S(O)₂R^(†), —S(O)₂NR^(†) ₂,—C(S)NR^(†) ₂, —C(NH)NR^(†) ₂, or —N(R^(†))S(O)₂R^(†); wherein eachR^(†) is independently hydrogen, C₁₋₆ aliphatic which may be substitutedas defined below, unsubstituted —OPh, or an unsubstituted 5-6-memberedsaturated, partially unsaturated, or aryl ring having 0-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, or,notwithstanding the definition above, two independent occurrences ofR^(†), taken together with their intervening atom(s) form anunsubstituted 3-12-membered saturated, partially unsaturated, or arylmono- or bicyclic ring having 0-4 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur. Suitable substituents on the aliphaticgroup of R^(†) are independently halogen, —R^(•), -(haloR^(•)), —OH,—OR^(•), —O(haloR^(•)), —CN, —C(O)OH, —C(O)OR^(•), —NH₂, —NHR^(•),—NR^(•) ₂, or —NO₂, wherein each R^(•) is unsubstituted or wherepreceded by “halo” is substituted only with one or more halogens, and isindependently C₁₋₄ aliphatic, —CH₂Ph, —O(CH₂)₀₋₁Ph, or a 5-6-memberedsaturated, partially unsaturated, or aryl ring having 0-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticulare, subcapsular,subarachnoid, intraspinal and intrasternal injection and infusion.

The phrases “systemic administration,” “administered systemically,”“peripheral administration” and “administered peripherally” as usedherein mean the administration of a compound, drug or other materialother than directly into the central nervous system, such that it entersthe patient's system and, thus, is subject to metabolism and other likeprocesses, for example, subcutaneous administration.

The term “enriched” as used herein refers to a mixture having anincreased proportion of one or more species. In some embodiments, themixture is “enriched” following a process that increases the proportionof one or more desired species in the mixture. In some embodiments, thedesired species comprise(s) greater than 10% of the mixture. In someembodiments, the desired species comprise(s) greater than 25% of themixture. In some embodiments, the desired species comprise(s) greaterthan 40% of the mixture. In some embodiments, the desired speciescomprise(s) greater than 60% of the mixture. In some embodiments, thedesired species comprise(s) greater than 75% of the mixture. In someembodiments, the desired species comprise(s) greater than 85% of themixture. In some embodiments, the desired species comprise(s) greaterthan 90% of the mixture. In some embodiments, the desired speciescomprise(s) greater than 95% of the mixture. Such proportions can bemeasured any number of ways, for example, as a molar ratio, volume tovolume, or weight to weight.

The term “pure” refers to compounds that are substantially free ofcompounds of related non-target structure or chemical precursors (whenchemically synthesized). This quality may be measured or expressed as“purity.” In some embodiments, a target compound has less than about30%, 20%, 10%, 5%, 2%, 1%, 0.5%, and 0.1% of non-target structures orchemical precursors. In certain embodiments, a pure compound of presentinvention is only one prosapogenin compound (i.e., separation of targetprosapogenin from other prosapogenin).

The term “carbohydrate” refers to a sugar or polymer of sugars. Theterms “saccharide”, “polysaccharide”, “carbohydrate”, and“oligosaccharide”, may be used interchangeably. Most carbohydrates arealdehydes or ketones with many hydroxyl groups, usually one on eachcarbon atom of the molecule. Carbohydrates generally have the molecularformula C_(n)H_(2n)O_(n). A carbohydrate may be a monosaccharide, adisaccharide, trisaccharide, oligosaccharide, or polysaccharide. Themost basic carbohydrate is a monosaccharide, such as glucose, sucrose,galactose, mannose, ribose, arabinose, xylose, and fructose.Disaccharides are two joined monosaccharides. Exemplary disaccharidesinclude sucrose, maltose, cellobiose, and lactose. Typically, anoligosaccharide includes between three and six monosaccharide units(e.g., raffinose, stachyose), and polysaccharides include six or moremonosaccharide units. Exemplary polysaccharides include starch,glycogen, and cellulose. Carbohydrates may contain modified saccharideunits such as 2′-deoxyribose wherein a hydroxyl group is removed,2′-fluororibose wherein a hydroxyl group is replace with a fluorine, orN-acetylglucosamine, a nitrogen-containing form of glucose. (e.g.,2′-fluororibose, deoxyribose, and hexose). Carbohydrates may exist inmany different forms, for example, conformers, cyclic forms, acyclicforms, stereoisomers, tautomers, anomers, and isomers.

Minimal Saponin Analogues

One aspect of the present application relates to minimal saponinanalogues (also referred to as “truncated saponins”) that do not containthe branched trisaccharide domain of the standard saponin molecule. Inone embodiment, the minimal saponin analogue is a compound having thechemical structure of formula (I),

or a pharmaceutically acceptable salt thereof, wherein

is a single or double bond;

W is methyl, —CHO, —CH₂OR^(x), or —C(O)R^(y);

V is hydrogen or —OR^(x);

Y is CH₂, —O—, —NR—, or —NH—;

wherein R is hydrogen, an optionally substituted group selected fromacyl, arylalkyl, 6-10 membered aryl, C1-12 aliphatic or C1-C12heteroaliphatic;

Z is hydrogen, a cyclic or acyclic, optionally substituted moietyselected from the group consisting of acyl, aliphatic, heteroaliphatic,aryl, arylalkyl, heterocyclyl, and heteroaryl; or Z comprises acarbohydrate;

each occurrence of R^(x) is independently hydrogen or an oxygenprotecting group selected from the group consisting of alkyl ethers,benzyl ethers, silyl ethers, acetals, ketals, esters, carbamates, andcarbonates;

R^(y) is —OH, —OR, or a carboxyl protecting group, wherein the carboxylprotecting group when taken with its attached carbonyl group, is anester, amide, or hydrazide.

In some embodiments, W is methyl, —CHO or —CH₂OH. In other embodiments,V is H or OH. In other embodiments, W is methyl, —CHO or —CH₂OH and V isH or OH. In other embodiments, W is methyl and V is OH. In otherembodiments, W is CH₂OH and V is OH. W is CHO and V is OH.

In some embodiments, the minimal saponin analogue having the structureof formula (I), wherein

is a single or double bond; W is C(O)R, CH₂OR or CH₂R, wherein R is H,or an optionally substituted group selected from acyl, arylalkyl, aryl,heteroaryl, aliphatic, heteroaliphatic, cycloaliphatic and heterocyclylgroups; V is H or OH; Y is O; Z is a linear oligosaccharide or anoptionally substituted group selected from the group consisting ofamine, amide, acyl, arylalkyl, aryl, heteroaryl, aliphatic,heteroaliphatic, cycloaliphatic and heterocyclyl groups.

Another aspect of the present application relates to a minimal saponinanalogue having the structure of formula (II),

or a pharmaceutically acceptable salt thereof, wherein W is C(O)R, CH₂ORor CH₂R, wherein R is H, or an optionally substituted group selectedfrom acyl, arylalkyl, aryl, heteroaryl, aliphatic, heteroaliphatic,cycloaliphatic and heterocyclyl groups; V is H or OH; X is CH₂R_(m),C(O)R_(m), CH₂OR_(m), CH₂R_(m), OR_(m), or NHR_(m), wherein R_(m) is H,or an optionally substituted group selected from acyl, arylalkyl, aryl,heteroaryl, aliphatic, heteroaliphatic, cycloaliphatic and heterocyclylgroups, and each occurrence of R_(n) is independently a hydrogen, amonosaccharide, a disaccharide or a trisaccharide.

In some embodiments, the minimal saponin analogue of the presentapplication is produced from a precursor having the structure of formula(III)

wherein: W is Me, —CHO, or —CH₂OH, and V is H or OH.

Method of Synthesis

Another aspect of the present application relates to a process forpreparing the minimal saponin analogues of the present application. Insome embodiments, the process includes the production of the precursorhaving the structure of formula (III). In some embodiments, theprecursor of formula (III) is produced with the following steps:

a) reacting a compound of formula (100) with a protecting group to forma compound of formula (101), wherein W is Me, CHO, CH₂OH, or CH₂OR_(p);wherein R_(p) is H or a suitable protecting group as necessary toachieve regioselectivity; V is H or OR_(P), and TES is a triethylsilylprotecting group;

b) reacting the compound of formula (101) with the compound of formula(102) to form the compound of formula (103), wherein Bn is a benzylprotecting group;

c) reacting the compound of formula (103) with a reducing agent to formthe compound of formula (104);

d) coupling the compound of formula (104) with compound of formula (105)in the presence of an activating agent to form the compound of formula(106), wherein Boc is a tert-butyloxycarbonyl protecting group;

e) deprotecting the compound of formula (106) to form the compound offormula (III).

In some embodiments, the protecting group is selected from the groupconsisting of methyl, methoxylmethyl (MOM), methylthiomethyl (MTM),t-butylthiomethyl, (phenyldimethylsilyl)methoxymethyl (SMOM),benzyloxymethyl (BOM), p-methoxybenzyloxymethyl (PMBM),(4-methoxyphenoxy)methyl (p-AOM), guaiacolmethyl (GUM), t-butoxymethyl,4-pentenyloxymethyl (POM), siloxymethyl, 2-methoxyethoxymethyl (MEM),2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl,2-(trimethylsilyl)ethoxymethyl (SEMOR), tetrahydropyranyl (THP),3-bromotetrahydropyranyl, tetrahydrothiopyranyl, 1-methoxycyclohexyl,4-methoxytetrahydropyranyl (MTHP), 4-methoxytetrahydrothiopyranyl,4-methoxytetrahydrothiopyranyl S,S-dioxide,1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl (CTMP),1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl,2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl,1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl,1-methyl-1-benzyloxyethyl, 1-methyl-1-benzyloxy-2-fluoroethyl,2,2,2-trichloroethyl, 2-trimethylsilylethyl, 2-(phenylselenyl)ethyl,t-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl,benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl,p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl,p-phenylbenzyl, 2-picolyl, 4-picolyl, 3-methyl-2-picolyl N-oxido,diphenylmethyl, p,p′-dinitrobenzhydryl, 5-dibenzosuberyl,triphenylmethyl, α-naphthyldiphenylmethyl,p-methoxyphenyldiphenylmethyl, di(p-methoxyphenyl)phenylmethyl,tri(p-methoxyphenyl)methyl, 4-(4′-bromophenacyloxyphenyl)diphenylmethyl,4,4′,4″-tris(4,5-dichlorophthalimidophenyl)methyl,4,4′,4″-tris(levulinoyloxyphenyl)methyl,4,4′,4″-tris(benzoyloxyphenyl)methyl,3-(imidazol-1-yl)bis(4′,4″-dimethoxyphenyl)methyl,1,1-bis(4-methoxyphenyl)-1′-pyrenylmethyl, 9-anthryl,9-(9-phenyl)xanthenyl, 9-(9-phenyl-10-oxo)anthryl,1,3-benzodithiolan-2-yl, benzisothiazolyl S,S-dioxido, trimethylsilyl(TMS), triethylsilyl (TES), triisopropylsilyl (TIPS),dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS),dimethylthexylsilyl, t-butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl(TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl,diphenylmethylsilyl (DPMS), t-butylmethoxyphenylsilyl (TBMPS), formate,benzoylformate, acetate, chloroacetate, dichloroacetate,trichloroacetate, trifluoroacetate, methoxyacetate,triphenylmethoxyacetate, phenoxyacetate, p-chlorophenoxyacetate,3-phenylpropionate, 4-oxopentanoate (levulinate),4,4-(ethylenedithio)pentanoate (levulinoyldithioacetal), pivaloate,adamantoate, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate,2,4,6-trimethylbenzoate (mesitoate), alkyl methyl carbonate,9-fluorenylmethyl carbonate (Fmoc), alkyl ethyl carbonate, alkyl2,2,2-trichloroethyl carbonate (Troc), 2-(trimethylsilyl)ethyl carbonate(TMSEC), 2-(phenylsulfonyl) ethyl carbonate (Psec),2-(triphenylphosphonio) ethyl carbonate (Peoc), alkyl isobutylcarbonate, alkyl vinyl carbonate alkyl allyl carbonate, alkylp-nitrophenyl carbonate, alkyl benzyl carbonate, alkyl p-methoxybenzylcarbonate, alkyl 3,4-dimethoxybenzyl carbonate, alkyl o-nitrobenzylcarbonate, alkyl p-nitrobenzyl carbonate, alkyl S-benzyl thiocarbonate,4-ethoxy-1-napththyl carbonate, methyl dithiocarbonate, 2-iodobenzoate,4-azidobutyrate, 4-nitro-4-methylpentanoate, o-(dibromomethyl)benzoate,2-formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl,4-(methylthiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoate,2,6-dichloro-4-methylphenoxyacetate,2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetate,2,4-bis(1,1-dimethylpropyl)phenoxyacetate, chlorodiphenylacetate,isobutyrate, monosuccinoate, (E)-2-methyl-2-butenoate,o-(methoxycarbonyl)benzoate, α-naphthoate, nitrate, alkylN,N,N′,N′-tetramethylphosphorodiamidate, alkyl N-phenylcarbamate,borate, dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate,sulfate, methanesulfonate (mesylate), benzylsulfonate, and tosylate(Ts). In another embodiment, the process wherein the oxygen protectinggroup is triethylsilyl (TES). In another embodiment, the process whereinthe reducing reagent is phenyl selenol.

In certain embodiments, the minimal saponin analogues have a purity of80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% orgreater, 97% or greater, 98% or greater, 99% or greater.

Vaccine Composition

Another aspect of the present application relates to a vaccinecomposition comprising an antigen and the minimal saponin analogue ofthe present application as an adjuvant. In some embodiments, the vaccinecomposition further comprises additional adjuvants.

The vaccine compositions of the present application are useful asvaccines to induce active immunity towards antigens in subjects. Anyanimal that may experience the beneficial effects of the compositions ofthe present invention within the scope of subjects that may be treated.In some embodiments, the subjects are mammals. In some embodiments, thesubjects are humans.

Most protein and glycoprotein antigens are poorly immunogenic ornon-immunogenic when administered alone. Strong adaptive immuneresponses to such antigens often requires the use of adjuvants. Immuneadjuvants are substances that, when administered to a subject, increasethe immune response to an antigen or enhance certain activities of cellsfrom the immune system. An adjuvant may also allow the use of a lowerdose of antigen to achieve a useful immune response in a subject.

Common adjuvants include alum, Freund's adjuvant (an oil-in-wateremulsion with dead mycobacteria), Freund's adjuvant with MDP (anoil-in-water emulsion with muramyldipeptide, MDP, a constituent ofmycobacteria), alum plus Bordetella pertussis (aluminum hydroxide gelwith killed B. pertussis). Such adjuvants are thought to act by delayingthe release of antigens and enhancing uptake by macrophages. Immunestimulatory complexes (ISCOMs) such as Quil-A (a Quillaja saponinextract) are open cage-like complexes typically with a diameter of about40 nm that are built up by cholesterol, lipid, immunogen, and saponin.ISCOMs deliver antigen to the cytosol, and have been demonstrated topromote antibody response and induction of T helper cell as well ascytotoxic T lymphocyte responses in a variety of experimental animalmodels.

The vaccines of the present invention may be used to confer resistanceto infection or cancer by either passive or active immunization. Whenthe vaccines of the present invention are used to confer resistancethrough active immunization, a vaccine of the present invention isadministered to an animal to elicit a protective immune response whicheither prevents or attenuates a proliferative or infectious disease.When the vaccines of the present invention are used to confer resistanceto infection through passive immunization, the vaccine is provided to ahost animal (e.g., human, dog, or mouse), and the antisera elicited bythis vaccine is recovered and directly provided to a recipient suspectedof having an infection or disease or exposed to a causative organism.

The present invention thus concerns and provides a means for preventingor attenuating a proliferative disease resulting from organisms or tumorcells which have antigens that are recognized and bound by antiseraproduced in response to the immunogenic polypeptides included invaccines of the present invention. As used herein, a vaccine is said toprevent or attenuate a disease if its administration to an animalresults either in the total or partial attenuation (i.e., suppression)of a symptom or condition of the disease, or in the total or partialimmunity of the animal to the disease.

The administration of the vaccine (or the antisera which it elicits) maybe for either a “prophylactic” or “therapeutic” purpose. When providedprophylactically, the vaccine(s) are provided in advance of any symptomsof proliferative disease. The prophylactic administration of thevaccine(s) serves to prevent or attenuate any subsequent presentation ofthe disease. When provided therapeutically, the vaccine(s) is providedupon or after the detection of symptoms which indicate that an animalmay be infected with a pathogen or have a certain cancer. Thetherapeutic administration of the vaccine(s) serves to attenuate anyactual disease presentation. Thus, the vaccines may be provided eitherprior to the onset of disease proliferation (so as to prevent orattenuate an anticipated infection or cancer) or after the initiation ofan actual proliferation.

Thus, in one aspect the present invention provides vaccines comprisingone or more bacterial, viral, protozoal, or tumor-related antigens incombination with one or more inventive compounds. In some embodiments,the vaccine comprises a single bacterial, viral, protozoal, ortumor-related antigen in combination with one inventive compound. Insome embodiments, the vaccine comprises two or more bacterial, viral,protozoal, or tumor-related antigens in combination with a singleinventive compound. In some embodiments, the vaccine comprises a two ormore bacterial, viral, protozoal, or tumor-related antigens incombination with two or more inventive compounds. In some embodiments,the vaccine comprises a single bacterial, viral, protozoal, ortumor-related antigens in combination with two or more inventivecompounds.

In some embodiments, one or more antigens of provided vaccines arebacterial antigens. In certain embodiments, the bacterial antigens areantigens associated with a bacterium selected from the group consistingof Helicobacter pylori, Chlamydia pneumoniae, Chlamydia trachomatis,Ureaplasma urealyticum, Mycoplasma pneumoniae, Staphylococcus spp.,Staphylococcus aureus, Streptococcus spp., Streptococcus pyogenes,Streptococcus pneumoniae, Streptococcus viridans, Enterococcus faecalis,Neisseria meningitidis, Neisseria gonorrhoeae, Bacillus anthracis,Salmonella spp., Salmonella typhi, Vibrio cholera, Pasteurella pestis,Pseudomonas aeruginosa, Campylobacter spp., Campylobacter jejuni,Clostridium spp., Clostridium difficile, Mycobacterium spp.,Mycobacterium tuberculosis, Treponema spp., Borrelia spp., Borreliaburgdorferi, Leptospria spp., Hemophilus ducreyi, Corynebacteriumdiphtheria, Bordetella pertussis, Bordetella parapertussis, Bordetellabronchiseptica, hemophilus influenza, Escherichia coli, Shigella spp.,Erlichia spp., Rickettsia spp. and combinations thereof.

In certain embodiments, one or more antigens of provided vaccines areviral-associated antigens. In certain embodiments, the viral-associatedantigens are antigens associated with a virus selected from the groupconsisting of influenza viruses, parainfluenza viruses, mumps virus,adenoviruses, respiratory syncytial virus, Epstein-Barr virus,rhinoviruses, polioviruses, coxsackieviruses, echo viruses, rubeolavirus, rubella virus, varicell-zoster virus, herpes viruses, herpessimplex virus, parvoviruses, cytomegalovirus, hepatitis viruses, humanpapillomavirus, alphaviruses, flaviviruses, bunyaviruses, rabies virus,arenaviruses, filoviruses, HIV 1, HIV 2, HTLV-1, HTLV-II, FeLV, bovineLV, FeIV, canine distemper virus, canine contagious hepatitis virus,feline calicivirus, feline rhinotracheitis virus, TGE virus, foot andmouth disease virus, and combinations thereof.

In certain embodiments, one or more antigens of provided vaccines aretumor-associated antigens. In some embodiments, the tumor-associatedantigens are antigens selected from the group consisting of killed tumorcells and lysates thereof, MAGE-1, MAGE-3 and peptide fragments thereof;human chorionic gonadotropin and peptide fragments thereof;carcinoembryonic antigen and peptide fragments thereof, alphafetoprotein and peptide fragments thereof; pancreatic oncofetal antigenand peptide fragments thereof; MUC-1 and peptide fragments thereof, CA125, CA 15-3, CA 19-9, CA 549, CA 195 and peptide fragments thereof;prostate-specific antigens and peptide fragments thereof;prostate-specific membrane antigen and peptide fragments thereof;squamous cell carcinoma antigen and peptide fragments thereof; ovariancancer antigen and peptide fragments thereof; pancreas cancer associatedantigen and peptide fragments thereof; Her1/neu and peptide fragmentsthereof; gp-100 and peptide fragments thereof; mutant K-ras proteins andpeptide fragments thereof; mutant p53 and peptide fragments thereof;truncated epidermal growth factor receptor, chimeric proteinp210^(BCR-ABL), KH-1, N3, GM1, GM2, GD2, GD3, Gb3, Globo-H, STn, Tn,Lewis^(x), Lewis^(y), TF; and mixtures thereof.

In certain embodiments, an antigen is covalently bound to a compound offormula (I). In some embodiments, an antigen is not covalently bound toa compound of formula (I).

One of ordinary skill in the art will appreciate that vaccines mayoptionally include a pharmaceutically acceptable excipient or carrier.Thus, according to another aspect, provided vaccines comprise one ormore antigens that are optionally conjugated to a pharmaceuticallyacceptable excipient or carrier. In some embodiments, said one or moreantigens are conjugated covalently to a pharmaceutically acceptableexcipient. In other embodiments, said one or more antigens arenon-covalently associated with a pharmaceutically acceptable excipient.

As described above, adjuvants may be used to increase the immuneresponse to an antigen. According to the invention, provided vaccinesmay be used invoke an immune response when administered to a subject. Incertain embodiments, an immune response to an antigen may be potentiatedby administering to a subject a provided vaccine in an effect amount topotentiate the immune response of said subject to said antigen.

As described above, provided compounds may be used in cancer vaccines asadjuvants in combination with tumor-associated antigens. In certainembodiments, said vaccines may be used in the treatment or prevention ofneoplasms. In certain embodiments, the neoplasm is a benign neoplasm. Inother embodiments, the neoplasm is a malignant neoplasm. Any cancer maybe treated using compounds of the invention with an antigen.

In certain embodiments, the malignancy is a hematological malignancy.Hematological malignancies are types of cancers that affect the blood,bone marrow, and/or lymph nodes. Examples of hematological malignanciesthat may be treated using compounds of formula (I) include, but are notlimited to, acute lymphoblastic leukemia (ALL), acute myelogenousleukemia (AML), chronic myelogenous leukemia (CML), chronic lymphocyticleukemia (CLL), hairy cell leukemia, Hodgkin's lymphoma, non-Hodgkin'slymphoma, cutaneous T-cell lymphoma (CTCL), peripheral T-cell lymphoma(PTCL), Mantle cell lymphoma, B-cell lymphoma, acute lymphoblastic Tcell leukemia (T-ALL), acute promyelocytic leukemia, and multiplemyeloma.

Other cancers besides hematological malignancies may also be treatedusing compounds of formula (I). In certain embodiments, the cancer is asolid tumor. Exemplary cancers that may be treated using compounds offormula (I) include colon cancer, lung cancer, bone cancer, pancreaticcancer, stomach cancer, esophageal cancer, skin cancer, brain cancer,liver cancer, ovarian cancer, cervical cancer, uterine cancer,testicular cancer, prostate cancer, bladder cancer, kidney cancer,neuroendocrine cancer, breast cancer, gastric cancer, eye cancer,gallbladder cancer, laryngeal cancer, oral cancer, penile cancer,glandular tumors, rectal cancer, small intestine cancer, sarcoma,carcinoma, melanoma, urethral cancer, vaginal cancer, to name but a few.

In certain embodiments, compounds and pharmaceutical compositions of thepresent invention can be employed in combination therapies, that is, thecompounds and pharmaceutical compositions can be administeredconcurrently with, prior to, or subsequent to, one or more other desiredtherapeutics or medical procedures. The particular combination oftherapies (therapeutics or procedures) to employ in a combinationregimen will take into account compatibility of the desired therapeuticsand/or procedures and the desired therapeutic effect to be achieved. Itwill also be appreciated that the therapies employed may achieve adesired effect for the same disorder (for example, an inventive compoundmay be administered concurrently with another antiproliferative agent),or they may achieve different effects (e.g., control of any adverseeffects).

For example, other therapies or anticancer agents that may be used incombination with the inventive anticancer agents of the presentinvention include surgery, radiotherapy (γ-radiation, neutron beamradiotherapy, electron beam radiotherapy, proton therapy, brachytherapy,and systemic radioactive isotopes, to name a few), endocrine therapy,biologic response modifiers (interferons, interleukins, and tumornecrosis factor (TNF) to name a few), hyperthermia and cryotherapy,agents to attenuate any adverse effects (e.g., antiemetics), and otherapproved chemotherapeutic drugs, including, but not limited to,alkylating drugs (mechlorethamine, chlorambucil, Cyclophosphamide,Melphalan, Ifosfamide), antimetabolites (Methotrexate), purineantagonists and pyrimidine antagonists (6-Mercaptopurine,5-Fluorouracil, Cytarabile, Gemcitabine), spindle poisons (Vinblastine,Vincristine, Vinorelbine, Paclitaxel), podophyllotoxins (Etoposide,Irinotecan, Topotecan), antibiotics (Doxorubicin, Bleomycin, Mitomycin),nitrosoureas (Carmustine, Lomustine), inorganic ions (Cisplatin,Carboplatin), enzymes (Asparaginase), and hormones (Tamoxifen,Leuprolide, Flutamide, and Megestrol), to name a few. Additionally, thepresent invention also encompasses the use of certain cytotoxic oranticancer agents currently in clinical trials and which may ultimatelybe approved by the FDA (including, but not limited to, epothilones andanalogues thereof and geldanamycins and analogues thereof). For a morecomprehensive discussion of updated cancer therapies see,www.nci.nih.gov, a list of the FDA approved oncology drugs atwww.fda.gov/cder/cancer/druglistframe.htm, and The Merck Manual,Seventeenth Ed. 1999, the entire contents of which are herebyincorporated by reference.

Another aspect of the present application relates to a methods forimmunizing a subject with the vaccine composition of the presentapplication.

Method for Enhancing the Effect of Other Drugs

Another aspect of the present application relates to methods forenhancing the effect of a cytotoxic drug, such as an anti-cancer drug,with a minimal saponin analogue of formula (I) or a salt thereof.Examples of the cytotoxic drugs include, but are not limited to,anti-cancer agents may include alkylating agents, such as bendamustine,busulfan, carmustine, chlorambucil, cyclophosphamide, dacarbazine,ifosfamide, melphalan, procarbazine, streptozocin, temozolomide;anti-tumor antibiotics, such as actinomycin D/dactinomycin, bleomycin,daunorubicin, doxorubicin, doxorubicin (pegylated liposomal),epirubicin, idarubicin, mitomycin, mitoxantrone; plantalkaloids/microtubule inhibitors, such as etoposide, docetaxel,irinotecan, paclitaxel, topotecan, vinblastine, vincristine,vinorelbine; anti-metabolites, such as asparaginase, capecitabine,cytarabine, 5-fluoro uracil, fludarabine, gemcitabine, methotrexate,pemetrexed, raltitrexed; DNA linking agents, such as carboplatin,cisplatin, oxaliplatin; bisphosphonates, such as clodronate, ibandronicacid, pamidronate, zolendronic acid; biological agents, such asalemtuzamab, BCG, bevacizumab, cetuximab, denosumab, erlotinib,gefitinib, imatinib, interferon, ipilimumab, lapatinib, panitumumab,rituximab, sunitinib, sorafenib, temsirolimus, trastuzumab;hormones/other, such as anastrozole, abiraterone, amifostine,bexarotene, bicalutamide, buserelin, cyproterone, degarelix, exemestane,flutamide, folinic acid, fulvestrant, goserelin, lanreotide,lenalidomide, letrozole, leuprorelin, medroxyprogesterone, megestrol,mesna, octreotide, stilboestrol, tamoxifen, thalidomide, triptorelin.

Another aspect of the present application relates to a pharmaceuticalcomposition comprising an effective amount of a minimal saponin analogueof formula (I) or a salt thereof, and a cytotoxic drug.

Method of Treatment

Another aspect of the present application relates a method of treatinginfectious disease in a subject comprising administering to the subjecta therapeutically effective amount of a compound of formula (I). In someembodiments, the infection is bacterial. In some embodiments, theinfection is viral. In some embodiments, the infection is protozoal. Insome embodiments, the subject is human.

Another aspect of the present application relates to a pharmaceuticalcomposition comprising an effective amount of a minimal saponin analogueof formula (I) or a salt thereof, and a pharmaceutically acceptablecarrier.

Formulations

The minimal saponin analogues of the present application may be combinedwith a pharmaceutically acceptable excipient to form a pharmaceuticalcomposition. In certain embodiments, the pharmaceutical compositionincludes a pharmaceutically acceptable amount of an inventive compound.The amount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will vary depending upon thehost being treated, and the particular mode of administration. Theamount of active ingredient that can be combined with a carrier materialto produce a single dosage form will generally be that amount of thecompound which produces a therapeutic effect. Generally, this amountwill range from about 1% to about 99% of active ingredient, preferablyfrom about 5% to about 70%, most preferably from about 10% to about 30%.

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically-acceptable antioxidants include: watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like;oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

Formulations of the present invention include those suitable for oral,nasal, topical (including buccal and sublingual), rectal, vaginal and/orparenteral administration. The formulations may conveniently bepresented in unit dosage form and may be prepared by any methods wellknown in the art of pharmacy. In certain embodiments, a formulation ofthe present invention comprises an excipient selected from the groupconsisting of cyclodextrins, liposomes, micelle forming agents, e.g.,bile acids, and polymeric carriers, e.g., polyesters and polyanhydrides;and a compound of the present invention. In certain embodiments, anaforementioned formulation renders orally bioavailable a compound of thepresent invention.

Formulations of the invention suitable for oral administration may be inthe form of capsules, cachets, pills, tablets, lozenges (using aflavored basis, usually sucrose and acacia or tragacanth), powders,granules, or as a solution or a suspension in an aqueous or non-aqueousliquid, or as an oil-in-water or water-in-oil liquid emulsion, or as anelixir or syrup, or as pastilles (using an inert base, such as gelatinand glycerin, or sucrose and acacia) and/or as mouth washes and thelike, each containing a predetermined amount of a compound of thepresent invention as an active ingredient. A compound of the presentinvention may also be administered as a bolus, electuary or paste.

In solid dosage forms of the invention for oral administration(capsules, tablets, pills, dragees, powders, granules and the like), theactive ingredient is mixed with one or more pharmaceutically-acceptablecarriers, such as sodium citrate or dicalcium phosphate, and/or any ofthe following: fillers or extenders, such as starches, lactose, sucrose,glucose, mannitol, and/or silicic acid; binders, such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,sucrose and/or acacia; humectants, such as glycerol; disintegratingagents, such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate; solutionretarding agents, such as paraffin; absorption accelerators, such asquaternary ammonium compounds; wetting agents, such as, for example,cetyl alcohol, glycerol monostearate, and non-ionic surfactants;absorbents, such as kaolin and bentonite clay; lubricants, such as talc,calcium stearate, magnesium stearate, solid polyethylene glycols, sodiumlauryl sulfate, and mixtures thereof; and coloring agents. In the caseof capsules, tablets and pills, the pharmaceutical compositions may alsocomprise buffering agents. Solid compositions of a similar type may alsobe employed as fillers in soft and hard-shelled gelatin capsules usingsuch excipients as lactose or milk sugars, as well as high molecularweight polyethylene glycols and the like.

Liquid dosage forms for oral administration of the compounds of theinvention include pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. In addition to the activeingredient, the liquid dosage forms may contain inert diluents commonlyused in the art, such as, for example, water or other solvents,solubilizing agents and emulsifiers, such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor and sesame oils),glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acidesters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active compounds, may contain suspendingagents as, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

Examples of suitable aqueous and nonaqueous carriers, which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofthe action of microorganisms upon the subject compounds may be ensuredby the inclusion of various antibacterial and antifungal agents, forexample, paraben, chlorobutanol, phenol sorbic acid, and the like. Itmay also be desirable to include isotonic agents, such as sugars, sodiumchloride, and the like into the compositions. In addition, prolongedabsorption of the injectable pharmaceutical form may be brought about bythe inclusion of agents which delay absorption such as aluminummonostearate and gelatin.

Injectable depot forms are made by forming microencapsule matrices ofthe subject compounds in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions, which are compatible with body tissue.

In certain embodiments, a compound or pharmaceutical preparation isadministered orally. In other embodiments, the compound orpharmaceutical preparation is administered intravenously. Alternativerouts of administration include sublingual, intramuscular, andtransdermal administrations.

The preparations of the present application may be given orally,parenterally, topically, or rectally. They are of course given in formssuitable for each administration route. For example, they areadministered in tablets or capsule form, by injection, inhalation, eyelotion, ointment, suppository, etc. administration by injection,infusion or inhalation; topical by lotion or ointment; and rectal bysuppositories. Oral administrations are preferred.

The minimal saponin analogues of the present application may beadministered to humans and other animals for therapy by any suitableroute of administration, including orally, nasally, as by, for example,a spray, rectally, intravaginally, parenterally, intracisternally andtopically, as by powders, ointments or drops, including buccally andsublingually.

Regardless of the route of administration selected, the minimal saponinanalogues of the present application, which may be used in a suitablehydrated form, and/or the pharmaceutical compositions of the presentinvention, are formulated into pharmaceutically-acceptable dosage formsby conventional methods known to those of skill in the art.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain an amountof the active ingredient that is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular compound of the presentinvention employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion ormetabolism of the particular compound being employed, the duration ofthe treatment, other drugs, compounds and/or materials used incombination with the particular compound employed, the age, sex, weight,condition, general health and prior medical history of the patient beingtreated, and like factors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, the physician or veterinarian couldstart doses of the compounds of the invention employed in thepharmaceutical composition at levels lower than that required to achievethe desired therapeutic effect and then gradually increasing the dosageuntil the desired effect is achieved.

In some embodiments, a compound or pharmaceutical composition of theapplication is provided to a subject chronically. Chronic treatmentsinclude any form of repeated administration for an extended period oftime, such as repeated administrations for one or more months, between amonth and a year, one or more years, or longer. In many embodiments, achronic treatment involves administering a compound or pharmaceuticalcomposition of the invention repeatedly over the life of the subject.Preferred chronic treatments involve regular administrations, forexample one or more times a day, one or more times a week, or one ormore times a month. In general, a suitable dose such as a daily dose ofa compound of the invention will be that amount of the compound that isthe lowest dose effective to produce a therapeutic effect. Such aneffective dose will generally depend upon the factors described above.Generally doses of the compounds of this invention for a patient, whenused for the indicated effects, will range from about 0.0001 to about100 mg per kg of body weight per day. Preferably the daily dosage willrange from 0.001 to 50 mg of compound per kg of body weight, and evenmore preferably from 0.01 to 10 mg of compound per kg of body weight.However, lower or higher doses can be used. In some embodiments, thedose administered to a subject may be modified as the physiology of thesubject changes due to age, disease progression, weight, or otherfactors.

In some embodiments, provided adjuvant compounds are administered aspharmaceutical compositions or vaccines. In certain embodiments, theamount of adjuvant compound administered is 1-2000 μg. In certainembodiments, the amount of adjuvant compound administered is 1-1000 μg.In certain embodiments, the amount of adjuvant compound administered is1-500 μg. In certain embodiments, the amount of adjuvant compoundadministered is 1-250 μg. In certain embodiments, the amount of adjuvantcompound administered is 100-1000 μg. In certain embodiments, the amountof adjuvant compound administered is 100-500 μg. In certain embodiments,the amount of adjuvant compound administered is 100-200 μg. In certainembodiments, the amount of adjuvant compound administered is 250-500 μg.In certain embodiments, the amount of adjuvant compound administered is10-1000 μg. In certain embodiments, the amount of adjuvant compoundadministered is 500-1000 μg. In certain embodiments, the amount ofadjuvant compound administered is 50-250 μg. In certain embodiments, theamount of adjuvant compound administered is 50-500 μg.

If desired, the effective daily dose of the active compound may beadministered as two, three, four, five, six or more sub-dosesadministered separately at appropriate intervals throughout the day,optionally, in unit dosage forms.

While it is possible for a compound of the present invention to beadministered alone, in certain embodiments the compound is administeredas a pharmaceutical formulation (composition) as described above.

The compounds according to the invention may be formulated foradministration in any convenient way for use in human or veterinarymedicine, by analogy with other pharmaceuticals.

The invention provides kits comprising pharmaceutical compositions of aninventive compound. In certain embodiments, such kits including thecombination of a compound of formula (I) and an antigen. The agents maybe packaged separately or together. The kit optionally includesinstructions for prescribing the medication. In certain embodiments, thekit includes multiple doses of each agent. The kit may includesufficient quantities of each component to treat a subject for a week,two weeks, three weeks, four weeks, or multiple months. The kit mayinclude a full cycle of immunotherapy. In some embodiments, the kitincludes a vaccine comprising one or more bacterial, viral, protozoal,or tumor-associated antigens, and one or more provided compounds.

The entire contents of all references cited above and herein are herebyincorporated by reference.

The description herein is for the purpose of teaching the person ofordinary skill in the art how to practice the present disclosure, and itis not intended to detail all those obvious modifications and variationsof it which will become apparent to the skilled worker upon reading thedescription. The specific embodiments of the present application havebeen presented for purposes of illustration and description. They arenot intended to be exhaustive or to limit the application and method ofuse to the precise forms disclosed. Obviously many modifications andvariations are possible in light of the above teaching. It is understoodthat various omissions or substitutions of equivalents are contemplatedas circumstance may suggest or render expedient, but is intended tocover the application or implementation without departing from thespirit or scope of the claims of the present application.

EXAMPLES Example 1 Initial Evaluation of Iodinated Saponins 6 and 8

A radioiodine (¹³¹I) was introduced into the QS-21 saponin scaffold toenable in vivo biodistribution studies. The non-radiolabeled aryl iodide6 (SQS-0-0-5-18) by acylation of amine 2 (SQS-0-0-5-11) was synthesized(FIG. 1b ). Because no in vitro model exists to assess adjuvantactivity, biological evaluation of aryl iodide 6 was carried out in apreclinical mouse vaccination model involving a multi-antigenformulation comprised of the immunogenic peptide MUC1 (prostate andbreast cancer antigen, non-glycosylated tandem repeat) conjugated to thehighly immunogenic KLH carrier protein (MUC1-KLH) and OVA, a reliableimmunogen that induces both antibody and T-cell responses in mice.Antibody responses against each of the three antigens, co-administeredwith the adjuvant of interest, were determined by ELISA.

Aryl iodide saponin 6 (SQS-0-0-5-18) induced antibody titers comparableto both NQS-21 (natural QS-21) and SQS-21 (synthetic QS-21, 65% 1a:35%1b) (FIG. 1d-f ) and also exhibited reduced toxicity compared to QS 21,as assessed by mouse weight loss (FIG. 1g ). As a negative control, theiodinated saponin variant 8 (SQS-0-3-7-18) was synthesized, which lacksthe linear tetrasaccharide domain (FIG. 1c and FIG. 7) and exhibitedpoor adjuvant activity (FIG. 7).

Reactions were performed in flame-dried sealed-tubes or modified Schlenk(Kjeldahl shape) flasks fitted with a glass stopper under a positivepressure of argon, unless otherwise noted. Air- and moisture-sensitiveliquids and solutions were transferred via syringe. The appropriatecarbohydrate reagents were dried via azeotropic removal of water withtoluene. Molecular sieves were activated at 350° C. and were crushedimmediately prior to use, then flame-dried under vacuum. Organicsolutions were concentrated by rotary evaporation below 30° C. Flashcolumn chromatography was performed employing 230-400 mesh silica gel.Thin-layer chromatography was performed using glass plates pre-coated toa depth of 0.25 mm with 230-400 mesh silica gel impregnated with afluorescent indicator (254 nm).

Example 2 Biodistribution of Radioiodinated Saponins [¹³¹I]⁻⁶ and[¹³¹I]⁻⁸

For biodistribution studies, radiolabeled aryl iodide saponin [¹³¹I]⁻⁶([¹³¹I]-SQS-0-0-5-18) was synthesized via aryl tin-halide exchange oftrimethylstannane 7 (FIG. 1b ). Radioiodinated [¹³¹I]⁻⁸([¹³¹I]-SQS-0-3-7-18) was synthesized analogously from 9 as a negativecontrol (FIG. 1c ).

To identify tissues and organs that could play roles in saponinmechanisms of action, the acute in vivo biodistribution of the activeadjuvant [¹³¹I]⁻⁶ was compared and the attenuated adjuvant [¹³¹I]⁻⁸ inmice co-administered with OVA. Treatment with [¹³¹I]⁻⁶, compared to[¹³¹I]⁻⁸, resulted in significantly higher recovery of radioactivity atthe site of injection (17-fold higher, 78% ID/g [injected dose pergram]) and in the nearest draining lymph nodes (24-fold higher, 27%ID/g) at 24 h post-injection (FIG. 2a ), which was retained at 72 h and96 h post-injection (FIG. 8). In contrast, radioactivity in othertissues where large fold-differences were initially observed (muscle,bone, skin) decreased rapidly at the later timepoints. Minimaldeiodination of [¹³¹I]⁻⁶, evidenced by low thyroid uptake (0.21% ID/g),was observed at 24 h (FIG. 2a ), although deiodination increased atlater time-points (FIG. 8). In contrast, the attenuated adjuvant[131I]-8 was recovered at significantly lower levels at the site ofinjection (4.5% ID/g) and nearest draining lymph nodes (1.1% ID/g) at 24h post-injection (FIG. 2a ) and was further cleared from both sites atlater time-points (FIG. 8). Taken together, these data indicate thatonly the active adjuvant 6 (SQS-0-0-5-18) localizes to and is retainedat the injection site and lymph nodes, while the attenuated adjuvant 8(SQS-0-3-7-18) is not.

The molecular mechanisms of the adjuvant activity of QS-21 and itsvariants remain poorly understood. It has been reported that QS-21stimulates mixed Th1/Th2 helper T cell responses, corresponding tocellular and humoral immunity respectively, including antigen-specificCD8+ cytotoxic T lymphocytes. There is some evidence to suggest thatQS-21 does not bind to Toll-like receptors 2 and 4 and that it does notoperate by a depot effect, in which the adjuvant increases the lifetimeof the antigen, extending its presentation to the immune system. It hasalso been suggested that Quillaja saponins may, by analogy to tucaresol,bind covalently to amino groups on T cell surface receptors via imineformation at the C4-aldehyde substituent, providing costimulation for Tcell activation.

Other adjuvants that contain mixtures of Quillaja saponins havepreviously been reported to affect the biodistribution of antigens,although it is not known whether biodistribution of the adjuvant isinfluenced by the presence or absence of the antigen. To investigatethese effects with these structurally-defined QS-21 variants, thebiodistribution of active adjuvant [¹³¹I]⁻⁶ and inactive adjuvant[¹³¹I]⁻⁸ in absence of OVA was assessed (FIG. 9). In both cases, thebiodistribution profiles were comparable to those observed in thepresence of OVA above (FIG. 8), indicating that the presence of theantigen OVA does not impact the biodistribution of these saponinadjuvants.

In a complementary experiment, the biodistribution of [¹³¹I]-OVA in thepresence and absence of active adjuvant 6 was examined (FIG. 10).Although comparable biodistribution profiles resulted, thyroid uptakewas also high, indicative of rapid deiodination of [¹³¹I]-OVA, which iscommonly observed for radioiodinated proteins. Thus, the influence of 6upon OVA antigen biodistribution could not be assessed effectively andan alternative experimental approach was required.

To address this problem, in vivo fluorescence imaging experiments withfluorescein-labeled active adjuvant 3 (SQS-0-0-5-12) andAlexa-647-conjugated OVA (OVA-A647) were conducted. At 24-hpost-injection, we observed retention of the fluorescent saponin at theinjection site (FIG. 2b ) and accumulation within the draining lymphnodes (left node in FIG. 2c ), consistent with the biodistributionresults above. Immunohistochemical analysis of dissected nodes indicatedsubnodal localization of adjuvant 3 to the cortex of the draininginguinal node. Flow cytometric analysis demonstrateddendritic-cell-specific internalization of 3 within the lymph nodes.Moreover, while OVA-A647 was observed at the site of injection in micetreated with both the active adjuvant 3 and an unlabeled, inactiveadjuvant 2 (SQS-0-0-5-11) (FIG. 2b and FIG. 11), it only localized tothe nearest draining lymph nodes when co-injected with the activeadjuvant 3 (FIG. 2c ). Overall, these data suggest a role for the activesaponin 3 in the trafficking of the OVA antigen by antigen-presentingcells to the draining lymph nodes, where the antigen is presented to theadaptive immune system.

Example 3 Truncated Saponin Lacking Branched Trisaccharide Domain (16)

The requisite triterpene cores were selectively silylated (TESOTf,2,6-lutidine) at hydroxyl groups to provide protected triterpenes havinga free C28-carboxylic acid. β-Selective Schmidt glycosylation (BF3.OEt2)with trisaccharide trichloroacetimidate donor 12 (Chea, E. K. et al.Synthesis and preclinical evaluation of QS-21 variants leading tosimplified vaccine adjuvants and mechanistic probes. J. Am. Chem. Soc.134, 13448-13457 (2012)) followed by reduction of the azide (PhSeH) gavethe corresponding glycosyl esters. Acylation of the amine with6-((t-butoxycarbonyl)-amino)hexanoic acid (14) (EtOCOCl, Et3N) andsubsequent global deprotection by hydrogenolysis (H2, Pd/C) and acidhydrolysis (TFA/H2O) afforded the fully deprotected saponins bearing thefree amine at the terminus of the acyl chain domain. Late-stageacylation of the amine with succinimidyl esters 4 or 5 gave thecorresponding aryl iodides 16, 18-22 or the relevant aryl tin congeners,respectively (FIG. 3a ).

Having established saponin variant 6 (SQS-0-0-5-18) as a potent adjuvantwith low toxicity, the role of the branched trisaccharide domain inadjuvant activity was investigated. Truncated saponin 16 (SQS-1-0-5-18),which lacks this entire domain, was synthesized from quillaic acid 1)and protected trisaccharide 12 (FIG. 3a ). Remarkably, truncated saponin16 elicited KLH and MUC1 antibody responses comparable to those ofparent aryl iodide 6, NQS-21, and SQS-21, and significantly higher thanthose of the no-adjuvant control, with the exception of anti-MUC1 titersat the lower dose (20 μg) (FIGS. 3b,3c ). Antibody titers against OVAwere also similar to those elicited by parent aryl iodide saponin 6 andconsiderably higher than those of the no-adjuvant control (FIG. 3d ).Moreover, truncated saponin 16 exhibited much lower toxicity than NQS-21and SQS-21, slightly lower than that of even the parent aryl iodide 6(FIG. 3e ). Thus, the entire branched trisaccharide domain is notrequired for adjuvant activity in the truncated saponin variant 16. Thisrepresents a major simplification of the saponin structure and providesa more favorable activity/toxicity profile than QS-21 itself.

Example 4 Saponins with Targeted Triterpene Domain Modifications (18-22)

Groups of five mice (C57BL/6J, female, 6-8 weeks of age) were vaccinatedwith a three-component vaccine consisting of MUC1-KLH (2.5 mg) and OVA(20 mg), or a four-component vaccine that also included GD3-KLH (5 mg).Antigens were co-administered with the adjuvant of interest (5, 20, or50 mg) or without adjuvant (no-adjuvant control) in phosphate bufferedsaline (PBS, 100 mL) via subcutaneous injections on days 0, 7, and 14,followed by a booster on day 65. Mouse sera were collected at day 72 andantibody titers against each antigen were determined by ELISA.Statistical significance of each antibody response compared to theno-adjuvant control was assessed using a two-tailed unpaired Student'st-test with CI=95%. As an initial, general assessment of toxicity, mouseweight loss was monitored on days 0, 1, 2, 3, and 7 after the firstvaccination. These animal experiments were conducted as described inMSKCC Institutional Animal Care and Use Committee (IACUC) protocol#97-11-051.

The aryl tin saponins (7, 9, 17, S9) were synthesized from thecorresponding amine precursors (2, S4, 15, S8) by acylation withN-succinimidyl-4-(trimethylstannyl)benzoate 4 (Et₃N, DMF, 21° C., 1-2.5h). Radiolabeling was achieved by iodination of the aryl tin saponin (20μg) with [¹³¹I]-NaI and chloramine-T (methanol, 21° C., 1 min), followedby immediate HPLC purification. Solvents were removed by rotaryevaporation at 35° C. and the radioiodinated probes were formulated in0.9% saline for biodistribution studies. Co-elution of theradioiodinated probes with the corresponding cold saponins was used forquality control analysis.

Radiolabeled ovalbumin was synthesized by treating 20 μg of ovalbuminwith [¹³¹I]-NaI and Chloramine-T in methanol. The reaction mixture wasdiluted with 2 mL phosphate buffered saline (PBS) followed bycentrifugal filtration at 2800 rpm for 12 min using a 30 kDa molecularweight cutoff filter. This process was repeated twice and theconcentrated compound was then formulated in 1 mL 0.9% saline forbiodistribution studies.

The discovery that the entire branched trisaccharide domain is notrequired for adjuvant activity facilitated investigation of thetriterpene domain of QS-21 by semisynthesis of new variants fromalternative triterpene precursors, by analogy to the synthesis of 16(SQS-1-0-5-18) from quillaic acid (FIG. 3a ). The roles of theC4-aldehyde substituent and C16-alcohol in the quillaic acid corestructure were of particular interest. Previously, the C4-aldehydesubstituent has been proposed to be important for the adjuvant activityof QS-21. However, other saponins that lack a triterpene aldehydesubstituent but are active adjuvants have been identified recently.Thus, an initial variant 18 (SQS-1-7-5-18) was synthesized fromoleanolic acid (FIG. 12), which shares the same carbon skeleton asquillaic acid, differing only in the oxidation states at the C4substituent (Me vs. CHO) and C16 (H vs. OH).

Oleanolic acid derivative 18 (C16-Me, C16-H) led to lower antibodytiters against all three antigens compared to the parent quillaic acidderivative 16 (FIG. 4a-d ). Further, OVA antibody titers with oleanolicacid derivative 18 were similar to those in the no-adjuvant control.Thus, removal of both the C4-aldehyde substituent and C16-alcohol inoleanolic acid derivative 18 results in considerably attenuated antibodyresponses in this preclinical vaccination model.

To investigate the importance of each of these functionalitiesindividually, triterpene variants 19-22, in which the oxidation statesof the C4-aldehyde substituent and C16-alcohol are varied independentlywas synthesized (FIG. 5a ). Caulophyllogenin variant 19 (SQS-1-11-5-18),in which the C4-aldehyde substituent is reduced to a hydroxymethyl groupwhile the C16-alcohol is retained, was accessed from an advancedintermediate in the synthesis of 16 (SQS-1-0-5-18) (FIG. 13).Echinocystic acid variant 20 (SQS-1-8-5-18), in which the C4-aldehydesubstituent is replaced by a methyl group while the C16-alcohol is againretained, and hederagenin variant 22 (SQS-1-10-5-18), in which theC4-aldehyde substituent is replaced by a hydroxymethyl group and theC16-alcohol is replaced by a proton, were prepared from thecorresponding, commercially available triterpenes (FIG. 14 and FIG. 15).Gypsogenin variant 21 (SQS-1-9-5-18), which possesses the C4 aldehydesubstituent but lacks the C16-alcohol, was accessed via initial TEMPOoxidation of the C4-hydroxymethyl substituent in hederagenin to affordgypsogenin.

These saponin variants were evaluated with a four-component vaccinecomprised of MUC1-KLH, OVA, and the poorly immunogenic glycolipid GD3(melanoma, neuroblastoma, sarcoma antigen) conjugated to KLH (GD3-KLH)(FIG. 5b-e ; see FIG. 16 for full data with both 20 and 50 μg doses).Echinocystic acid derivative 20 (C4-Me, C16-OH), induced the highestantibody responses to all four antigens, comparable to or higher thanthose of the complete, branched trisaccharide-containing saponin 6 andSQS-21. Caulophyllogenin derivative 19 (C4-CH2OH, C16-OH) affordedantibody titers slightly below those of echinocystic acid derivative 20,branched trisaccharide-containing saponin 6, and SQS-21, albeit only atelevated doses (50 μg). In contrast, gypsogenin derivative 21 (C4-CHO,C16-H) and hederagenin derivative 22 (C4-CH2OH, C16-H), both generatedlower antibody responses in all cases except the anti-GD3 IgG response,and similar to the no-adjuvant treated controls for KLH and OVA.

Antibody subtyping of the anti-MUC1 and anti-OVA IgG isotypes revealed asignificant bias toward the mouse IgG1 and IgG2b subtype with both ofthe adjuvant active saponins in this group (19, 20). Similar resultswere obtained with SQS-21 and with the parent quillaic acid derivative16. Production of other mouse IgG subtypes, including IgG2a and IgG3,was low or negligible, as indicated by class-specific ELISA. Toxicityremained drastically lower for all four variants compared to NQS-21 andSQS-21 (FIG. 5f ).

Thus, echinocystic acid derivative 20 provides immunostimulatoryactivity generally rivaling that of SQS-21 but without the associatedtoxicity. Caulophyllogenin derivative 19 also provides antibodyresponses similar to the complete, branched trisaccharide-containingsaponin 6, although higher doses are required. Importantly, bothechinocystic acid derivative 20 and caulophyllogenin derivative 19 lackthe C4-aldehyde substituent but retain the C16-alcohol. In contrast,gypsogenin derivative 21 and hederagenin derivative 22 both lack theC16-alcohol and induced lower antibody responses to all antigens tested.Taken together, these data indicate that the C4-aldehyde substituent isnot required for potent immunoadjuvant activity in these novel saponinsand reveal a previously unappreciated role for the C16-alcohol inenhancing activity.

Example 5 Biodistribution of Truncated Saponins [¹³¹I]⁻¹⁶ and [¹³¹I]⁻¹⁸

Groups of five mice (naive, C57BL/6J female, 8-10 weeks of age) wereinjected subcutaneously with the radioiodinated saponin adjuvants ofinterest (˜25 mCi), the corresponding non-radiolabeled saponin (20 mg),and OVA (20 mg) in PBS (150 □L). Mice were sacrificed at 24 h, 72 h, and96 h post-injection. Tissues and organs were harvested and analyzed fordistribution of radioactivity normalized to the weight of the organ (%ID/g, percent injected dose per gram). Statistical significance of thedifference in recoveries (% ID/g) for the active and attenuated adjuvantwas assessed for each tissue or organ using two-tailed unpairedStudent's t-test with CI=95%. In initial experiments, biodistributionprofiles did not change substantially between 24 h and 96 hpost-injection, and the 24 h time-point was used for the subsequentexperiments.

Three mice were shaved and immunized in the left flank with 10 μg ofactive adjuvant 3 (SQS-0-0-5-12) or inactive adjuvant 2 (SQS-0-0-5-11)and 20 μg of Alexa-647-conjugated OVA (OVA-A647) in PBS (100 μL).Whole-body imaging was performed at 24 h post-injection with a MaestroImaging System. At 24 h post-injection, mice were sacrificed and theleft and right lymph nodes were dissected and imaged separately.

The in vivo biodistribution patterns of the adjuvant-active truncatedquillaic acid variant 16 (SQS-1-0-5-18) were compared and theadjuvant-attenuated oleanolic acid derivative 18 (SQS-1-7-5-18), usingthe radioiodinated congeners [¹³¹I]⁻¹⁶ (FIG. 3a ) and [¹³¹I]⁻¹⁸ (FIG.12), to enable correlation with the earlier studies of theactive/attenuated adjuvant pair 6 (SQS-0-0-5-18) and 8 (SQS-0-3-7-18)(FIG. 2a ). The active adjuvant [¹³¹I]⁻¹⁶ showed significantly higherlocalization at the injection site (136% ID/g) and within the lymphnodes (3.55% ID/g) compared to [¹³¹I]⁻¹⁸ (11.5% and 0.50% ID/g,respectively) (FIG. 6). Accordingly, in both biodistribution studies,the more active adjuvant was preferentially retained at the site ofinjection and accumulated in the lymph nodes, providing a positivecorrelation between this biodistribution pattern and adjuvant activity.

Example 7 Synthesis of Iodinated and Radiolabeled Saponin Adjuvants

Synthesis of Initial Variants 6 (SQS-0-0-5-18) and 8 (SQS-0-3-7-18)

SQS-0-0-5-18 (6).

(EC-V-056) To a solution of amine 2 (see Chea, E. K. et al. Synthesisand preclinical evaluation of QS-21 variants leading to simplifiedvaccine adjuvants and mechanistic probes. J. Am. Chem. Soc. 134,13448-13457 (2012)) (9.0 mg, 6.0 μmol, 1.0 equiv) inN,N′-dimethylformamide (2.0 mL), triethylamine (50 μL, 0.36 mmol, 60equiv) was injected and the mixture stirred at 21° C. for 50 min. Aryliodide 4 (see (a) Zhi, Y.-G. et al. Systematic studies onphotoluminescence of oligo(arylene-ethynylene)s: tunability of excitedstates and derivatization as luminescent labeling probes for proteins.Eur. J. Org. Chem. 3125-3139 (2006); (b) Shell, T. A., Mohler, D. L.Selective targeting of DNA for cleavage within DNA-histone assemblies bya spermine-[CpW(CO)₃Ph]₂ conjugate. Org. Biomol. Chem. 3, 3091-3093(2005)) (20 mg, 60 gmol, 10 equiv) in N,N′-dimethylformamide (0.6 mL)was then added dropwise and the reaction stirred at 21° C. for 1 h. Thecontents were diluted with 20% acetonitrile/water (10 mL) and directlypurified by RP-HPLC on an XBridge Prep BEH300 C18 column (5 μm, 10×250mm) using a linear gradient of 20-70% acetonitrile/water, over 30 min,at a flow rate of 5 mL/min. SQS-0-0-5-18 (6) (5.4 mg, 52% yield) wasobtained as a white powder after lyophilization.

Aryl Tin Precursor to [¹³¹I]-SQS-0-0-5-18 (7).

(EC-V-052) To a solution of amine 2 (2.0 mg, 1.3 μmol, 1.0 equiv) inN,N′-dimethylformamide (0.9 mL) triethylamine (10 μL, 72 gmol, 55 equiv)was injected and the mixture stirred at 21° C. for 50 min. Aryl tin 5(Koziorowski, J., Henssen, C., Weinreich, R. A new convenient route toradioiodinated N-succinimidyl 3- and 4-iodobenzoate, two reagents forradioiodination of proteins. Appl. Radiat. Isot. 49, 955-959 (1998))(2.0 mg, 5.2 μmol, 4.0 equiv) in N,N′-dimethylformamide (0.2 mL) wasthen added dropwise and the reaction stirred at 21° C. for 1 h. Afterthis time, the contents were diluted with 20% acetonitrile/water (10mL), and directly purified by RP-HPLC on an XBridge Prep BEH300 C18column (5 μm, 10×250 mm) using a 20-70% acetonitrile/water lineargradient, over 30 min, at a flow rate of 5 mL/min. Saponin 7 (1.8 mg,78% yield) was obtained as a white powder after lyophilization.

Fully Protected Aminoacyl Prosapogenin S3.

(EC-V-191) A solution of acid S1 (Deng, K., Adams, M. M., Gin, D. Y.Synthesis and structure verification of the vaccine adjuvant QS-7-Api.Synthetic access to homogeneous Quillaja saponaria immunostimulants. J.Am. Chem. Soc. 130, 5860-5861 (2008)) (50 mg, 24 mol, 1.0 equiv) indichloromethane (1.56 mL) and pyridine (40 μL, 0.50 mmol, 20.5 equiv)was cooled in an ice bath. After stirring for 5 min, thionyl chloride(20 μL, 0.28 mmol, 11.5 equiv) was injected followed by addition ofN,N′-dimethylformamide (6.25 μL, 0.081 mmol, 3.4 equiv) and stirred at21° C. for 1.5 h. The resulting clear-yellow solution was concentratedto afford an amorphous white solid that was then redissolved indichloromethane (1.6 mL) containing pyridine (40 μL, 0.50 mmol, 20.5equiv). To the solution was injected S2 (0.1 mL, 0.62 mmol, 25.8 equiv),which caused an orange tint to form. After 30 min, the reaction wasdiluted with CH₂Cl₂ (30 mL) and washed with saturated sodium bicarbonate(30 mL). The aqueous phase was extracted with CH₂Cl₂ (2×30 mL) and thecombined organic phases were dried over Na₂SO₄, filtered, and evaporatedto dryness to give a bright yellow oil. Purification by silica gelchromatography (4:1 hexanes/EtOAc) afforded S3 (48 mg, 91% yield) as aglassy solid.

Aminoacyl Prosapogenin S4.

(EC-IV-187) In a 10 mL round-bottom flask, S3 (24 mg, 10.8 μmol, 1.0equiv) was dissolved in tetrahydrofuran/ethanol (5 mL, 1:1) and 10% (drybasis) palladium on carbon, wet, Degussa type E101 NE/W (63 mg, 29.6μmol, 2.7 equiv) was added. The reaction was stirred under hydrogenpressure (50 psi) at 21° C. for 24 h. The resulting crude mixture ofpartially desilylated products was filtered through a 0.45 μm nylonsyringe filter, rinsed with methanol (20 mL), CH₂Cl₂ (10 mL), andmethanol again (5 mL), and the clear filtrate was evaporated to dryness.Successful debenzylation is assessed by the disappearance of aromaticresonances by ¹H NMR in CD₃OD. The resulting mixture was then subjectedtrifluoroacetic acid/water (2 mL, 4:1) for 3.3 h in an ice bath and thenevaporated to dryness to afford a pink solid. The crude obtained waspurified by RP-HPLC on an XBridge Prep BEH300 C18 column (5 μm, 10×250mm) using a linear gradient of 20-95% acetonitrile/water (0.05% TFA),over 20 min, at a flow rate of 5 mL/min. Saponin S4 (5.4 mg, 50% yield)eluted as a broad single peak and existed as a white powder afterlyophilization.

SQS-0-3-7-18 (8).

(EC-IV-194) To a solution of S4 (7.1 mg, 7.1 μmol, 1.0 equiv) inN,N′-dimethylformamide (0.4 mL) was injected triethylamine (20 pt, 0.14mmol, 20 equiv), followed by dropwise addition of 4 (14 mg, 40.6 μmol,5.7 equiv) in N,N′-dimethylformamide (0.4 mL). After stirring for 3 h,the contents were diluted with 10 mL water (0.05% TFA) and purified byRP-HPLC on an XBridge Prep BEH300 C18 column (5 μm, 10×250 mm) using alinear gradient of 30-80% acetonitrile/water (0.05% TFA), over 30 min,at a flow rate of 5 mL/min. SQS-0-3-7-18 (8) (5.9 mg, 68% yield) elutedas a single peak and existed as a white powder after lyophilization.

Aryl Tin Precursor to [¹³¹I]-SQS-0-3-7-18 (9).

(EC-IV-193) To S4 (3.6 mg, 3.6 μmol, 1.0 equiv) dissolved inN,N′-dimethylformamide (0.2 mL) with triethylamine (20 μL, 0.14 mmol, 40equiv) was added dropwise a solution of 5 (5 mg, 13.1 gmol, 3.6 equiv)in N,N′-dimethylformamide (0.1 mL). After stirring for 2.5 h, thecontents were diluted with water (4 mL) and purified via RP-HPLC on anXBridge Prep BEH300 C18 column (5 μm, 10×250 mm) using a 20-95%acetonitrile/water linear gradient, over 30 min, at a flow rate of 5mL/min. Saponin 9 (2.4 mg, 53% yield) eluted as a single peak and wasobtained as a white powder after lyophilization.

Synthesis of Variant Lacking the Branched Trisaccharide Domain (16)

Bis(silyl ether) of Quillaic Acid (11).

(AFT-II-040) A suspension of quillaic acid 10 (200 mg, 0.41 mmol, 1.0equiv) in CH₂Cl₂ (20 mL) was cooled in an ice bath and 2,6-lutidine(0.48 mL, 4.1 mmol, 10 equiv) and triethylsilyltrifluoromethanesulfonate (0.46 mL, 2.06 mmol, 5.0 equiv) were injected.After stirring for 1 h, the contents were washed with saturated NaHCO₃(10 mL), the aqueous phase was extracted with CH₂Cl₂ (2×15 mL) and thecombined organics were dried over Na₂SO₄, filtered, and concentrated.The crude product was purified by silica gel chromatography (hexanes to4:1 hexanes/EtOAc) to afford 11 (235 mg, 80% yield).

Protected Quillaic Acid Saponin Azide S28.

(AFT-I-165) To a solution of 11 (38 mg, 49 gmol, 1.05 equiv) and imidate12 (52 mg, 47 μmol, 1.0 equiv) in CH₂Cl₂ (7 mL) 80 mg powdered 4 Åmolecular sieves was added and the mixture was stirred at 21° C. for 30min. The reaction schlenk was then cooled to −35° C. and borontrifluoride diethyletherate (1.2 μL, 9.0 μmol, 0.2 equiv) was injected.The mixture was stirred for 0.5 h at this temperature, quenched with 0.2mL of triethylamine and concentrated. Purification of the residue bysilica gel chromatography (0.2% triethylamine in benzene to 97:3benzene/EtOAc) gave a colorless oil that was further chromatographed toafford the desired product S28 (56 mg, 72% yield) as a white solid.

Protected Quillaic Acid Saponin Amine 13.

(AFT-I-167) To S28 (62 mg, 37 μmol, 1.0 equiv) dissolved intriethylamine (28 mL) was added a freshly prepared solution of phenylselenol (1.11 mmol, 30 equiv) via cannula. Upon addition of phenylselenol a white precipitate was formed and the solution became brightyellow. The reaction was stirred for 8 h at 38° C. and the solution wasthen concentrated to afford a yellow-white solid. The crude mixture waspurified by silica gel chromatography (90:10 to 85:15 benzene/EtOAc toafford the amine 13 (49 mg, 80% yield) as a glassy solid.

Fully Protected Aminoacyl Quillaic Acid Saponin S10.

(AFT-I-169) To a clear, colorless solution of6-((t-butoxycarbonyl)-amino)hexanoic acid (14) (45 mg, 0.20 mmol, 11.5equiv) in tetrahydrofuran (2.5 mL) at 0° C. was added triethylamine (213μL, 1.53 mmol, 90 equiv) followed by ethyl chloroformate (16.0 μL, 0.17mmol, 10.0 equiv). The turbid, white solution was stirred for 2.5 h at0° C. and then added via cannula to amine 13 (28 mg, 17.0 μmol, 1.0equiv) at 0° C. The reaction mixture was stirred at this temperature for1.5 h and then quenched with water (0.2 mL) to give a clear solution.The contents were diluted with saturated NaHCO₃ (30 mL), and the aqueousphase was extracted with CH₂Cl₂ (3×25 mL). The combined organics weredried (Na₂SO₄), filtered, and evaporated to dryness. Purification bysilica gel chromatography (2:1 hexanes/EtOAc with 0.2% triethylamine)afforded S10 (28 mg, 88% yield) as a white glassy solid.

Aminoacyl Quillaic Acid Saponin 15.

(AFT-I-204) In a 50 mL round-bottom flask, S10 (68 mg, 36.6 μmol, 1.0equiv) was dissolved in tetrahydrofuran/ethanol (20 mL, 1:1) and 10%(dry basis) palladium on carbon, wet, Degussa type E101 NE/W (390 mg,0.18 mmol, 5.0 equiv) was added. The reaction was stirred under hydrogenpressure (50 psi) at 21° C. for 24 h, and the suspension was filteredthrough a 0.45 μm nylon syringe filter, washed with methanol (3×30 mL)and concentrated. Successful debenzylation is assessed by thedisappearance of aromatic resonances by ¹H NMR in CD₃OD. The crudemixture was then dissolved in a solution of trifluoroacetic acid (8 mL,TFA/H₂O 3:1) and stirred for 2 h in an ice bath. The reaction wasevaporated to dryness to afford a white solid that was dissolved in 20%acetonitrile/water (20 mL) and purified via RP-HPLC on an XBridge PrepBEH300 C18 column (5 μm, 10×250 mm) using a linear gradient of 30-70%acetonitrile/water (0.05% TFA), over 15 min, at a flow rate of 5 mL/min.The aminoacyl quillaic acid saponin 15 eluted as a single peak and wasobtained as a white powder (28 mg, 74% yield) after lyophilization.

SQS-1-0-5-18 (16).

(AFT-I-300) To a solution of 15 (2.1 mg, 2.0 μmol, 1.0 equiv) inN,N′-dimethylformamide (0.4 mL) was added triethylamine (11 μL, 0.08mmol, 40 equiv) followed by dropwise addition of 4 (4.0 mg, 10 μmol, 5.8equiv) in N,N′-dimethylformamide (0.2 mL). After stirring for 2 h, thecontents were diluted with 30% acetonitrile/water (2.3 mL) and purifiedby RP-HPLC on an XBridge Prep BEH300 C18 column (5 μm, 10×250 mm) usinga linear gradient of 30-70% acetonitrile/water (0.05% TFA), over 15 min,at a flow rate of 5 mL/min. SQS-1-0-5-18 (16) (1.7 mg, 67% yield) wasobtained as a white powder after lyophilization.

Aryl Tin Precursor to [¹³¹I]-SQS-1-0-5-18 (17).

(EC-V-227) To 15 (0.65 mg, 0.63 μmol, 1.0 equiv) dissolved inN,N′-dimethylformamide (0.2 mL) was added triethylamine (10 μL, 72 μmol,114 equiv) and 5 (1.0 mg, 2.6 μmol, 4.1 equiv). After stirring for 1.5 hthe reaction was diluted with water (4 mL) and purified via RP-HPLC onan XBridge Prep BEH300 C18 column (5 μm, 10×250 mm) with a 35-95%acetonitrile/water linear gradient, over 30 min, at a flow rate of 5mL/min. Saponin 17 (0.6 mg, 75% yield) eluted as a single peak and wasobtained as a white powder after lyophilization.

Synthesis of Variants with Modifications in the Triterpene Domain(18-22)

Silyl Ether of Oleanolic Acid (S29).

(AFT-I-137) To a solution of oleanolic acid S5 (250 mg, 0.55 mmol, 1.0equiv) in CH₂Cl₂ (10 mL) at 0° C., 2,6-lutidine (0.38 mL, 3.28 mmol, 6.0equiv), and triethylsilyl trifluoromethanesulfonate (0.37 mL, 1.64 mmol,3.0 equiv) were added and the mixture was stirred for 1 h. The contentswere quenched with 0.5 N HCl (10 mL), and the aqueous phase wasextracted with CH₂Cl₂ (2×15 mL). The combined organics were dried overNa₂SO₄, filtered, concentrated and finally purified by silica gelchromatography (hexanes to 4:1 hexanes/EtOAc) to afford S29 (250 mg, 80%yield).

Protected Oleanolic Acid Saponin Azide S6.

(EC-V-215) To a solution of S29 (50 mg, 87 μmol, 1.0 equiv) and imidate12 (97 mg, 87 μmol, 1.0 equiv) in CH₂Cl₂ (8.0 mL) was added 120 mgpowdered 4 Å molecular sieves and the mixture was stirred at 21° C. for1 h. The reaction schlenk was then transferred to a −78° C. bath andboron trifluoride diethyletherate (8.8 μL, 70 μmol, 0.8 equiv) wasinjected. The reaction was stirred at −50° C. for 20 min, at 21° C. for1 min, cooled back to −50° C., stirred for 20 min and finally again at21° C. for 1 min. The mixture was then quenched with triethylamine (0.1mL) at −50° C. and passed through a plug of silica gel. The resultingfiltrate was concentrated, and purified by silica gel chromatography(hexanes to 5:1 hexanes/EtOAc) to afford S6 (89 mg, 68% yield) as aglassy solid.

Protected Oleanolic Acid Saponin Amine S30.

(EC-V-216) To S6 (44 mg, 29 μmol, 1.0 equiv) dissolved in triethylamine(12 mL) was added a freshly prepared solution of phenyl selenol (0.44mmol, 15 equiv) via cannula transfer. Upon addition of phenyl selenol awhite precipitate was formed and the mixture became bright yellow. Afterstirring at 38° C. for 8 h, the solution was concentrated to give ayellow-white solid, which was purified by silica gel chromatography (5:1hexanes/EtOAc to 2% triethylamine in EtOAc) to afford S30 (41 mg, 95%yield) as a white solid.

Fully Protected Aminoacyl Oleanolic Acid Saponin S7.

(EC-V-217) To a solution of 14 (63 mg, 0.27 mmol, 10 equiv) intetrahydrofuran (2.6 mL) was added triethylamine (365 μL, 2.6 mmol, 96equiv) at 0° C. To the clear, colorless solution was injected ethylchloroformate (23 μL, 025 mmol, 9.0 equiv), which turned the solutionturbid white. The acid activation was allowed to proceed at 0° C. for2.5 h before the entire solution was cannula transferred into a schlenckcontaining amine S30 (41 mg, 27 μmol, 1.0 equiv). The reaction mixturewas stirred at 0° C. for 1.5 h and then quenched with water (90 μL), atwhich point the solution turned from turbid, white to clear. Thecontents were then evaporated to dryness and purified by silica gelchromatography (5:1 hexanes/EtOAc) to afford S7 (40 mg, 81% yield) as awhite glassy solid.

Aminoacyl Oleanolic Acid Saponin S8.

(EC-V-218) In a 25 mL round-bottom flask containing S7 (10 mg, 5.8 μmol,1.0 equiv) was added tetrahydrofuran/ethanol (2 mL, 1:1) followed by 10%(dry basis) palladium on carbon, wet, Degussa type E101 NE/W (14.0 mg,6.5 μmol, 1.1 equiv). The reaction was stirred under hydrogen pressure(50 psi) at 21° C. for 24 h and then filtered through a 0.45 mm nylonsyringe filter, washed with methanol (20 mL), CH₂Cl₂ (10 mL), andmethanol again (5 mL) to thoroughly wash the palladium. The clearfiltrate was evaporated to dryness. Successful debenzylation wasassessed by the disappearance of aromatic resonances by ¹H NMR in CD₃OD.The mixture was then dissolved in a solution of trifluoroaceticacid/water (2 mL, 4:1) and stirred for 2 h in an ice bath. After thistime, the reaction was evaporated to dryness to give a white solid thatwas purified by RP-HPLC using a 30-80% acetonitrile/water (0.1% TFA)linear gradient, over 20 min, at a flow rate of 5 ml/min. The desiredproduct S8 (2.6 mg, 44% yield) was obtained as a white powder afterlyophilization.

SQS-1-7-5-18 (18).

(EC-V-221) Amine S8 (1.3 mg, 1.3 μmol, 1.0 equiv) was dissolved inN,N′-dimethylformamide (0.2 mL) and triethylamine (3.6 μL, 25.6 μmol, 20equiv) was injected. To this solution, 4 (2.5 mg, 7.3 μmol, 5.7 equiv)was added and the reaction mixture was stirred at 21° C. for 2 h. Afterthis time, the contents were diluted with 3 mL water (0.05% TFA) anddirectly purified by HPLC using a 30-80% acetonitrile/water (0.05% TFA)linear gradient, over 20 min, at a flow rate of 5 mL/min to affordSQS-1-7-5-18 (18) (1.0 mg, 63% yield) as a white powder afterlyophilization.

Aryl Tin Precursor to SQS-1-7-5-18 (S9).

(EC-V-222) To a solution of amine S8 (1.3 mg, 1.3 mol, 1.0 equiv) inN,N′-dimethylformamide (0.2 mL) triethylamine (3.6 μL, 25.6 μmol, 20equiv) was added followed by 5 (2.6 mg, 6.8 μmol, 5.2 equiv). Afterstirring at 21° C. for 1.5 h, the reaction was diluted with 3 mL waterand directly purified by RP-HPLC using a linear gradient of 30-90%acetonitrile/water, over 30 min, at a flow rate of 5 mL/min to afford S9(1.0 mg, 62% yield) as a white powder after lyophilization.

Fully Protected Aminoacyl Caullophylogenin Saponin S11.

(AFT-I-243). To a solution of S10 (10 mg, 5.4 μmol) in methanol (1.0 mL)NaBH₄ was added, and the reaction was stirred at 21° C. for 3 h. Themixture was then diluted with acetone (2 mL), concentrated, and purifiedby silica gel chromatography (85:15 benzene/EtOAc) to afford 511 (10mg, >99% yield).

Aminoacyl Caullophylogenin Saponin S12.

(AFT-I-244) In a 25 mL round-bottom flask, S11 (15 mg, 8.1 μmol, 1.0equiv) was dissolved in 4.0 mL tetrahydrofuran/ethanol (1:1) and 10%(dry basis) palladium on carbon, wet, Degussa type E101 NE/W (85 mg,0.04 mmol, 5.0 equiv) was added. The reaction was stirred under hydrogenatmosphere (balloon) at 21° C. for 12 h, and the suspension was filteredthrough a 0.45 μm nylon syringe filter, thoroughly washed with methanol(4×20 mL) and concentrated. Successful debenzylation is assessed by thedisappearance of aromatic resonances by ¹H NMR in CD₃OD. The crudemixture was then dissolved in a pre-cooled (0° C.) solution oftrifluoroacetic acid (3.2 mL, TFA/H₂O 3:1) and stirred at 0° C. for 1.25h. The reaction was evaporated to dryness, and the crude product wasdissolved in 20% acetonitrile/water (8 mL) and purified via RP-HPLC onan XBridge Prep BEH300 C18 column (5 μm, 10×250 mm) using a lineargradient of 20-70% acetonitrile/water (0.05% TFA), over 20 min, at aflow rate of 5 mL/min. The desired product S12 was obtained as a whitepowder (5.8 mg, 70% yield) after lyophilization.

SQS-1-11-5-18 (19).

(AFT-I-245) To a solution of S12 (7.0 mg, 6.7 μmol, 1.0 equiv) inN,N′-dimethylformamide (1.3 mL) was injected triethylamine (20 μL, 0.13mmol, 20 equiv) followed by dropwise addition of 4 (11.6 mg, 33.6 μmol,5.0 equiv) in N,N′-dimethylformamide (0.7 mL). After stirring for 3 h,the contents were diluted with 25% acetonitrile/water (10 mL) andpurified by RP-HPLC on an XBridge Prep BEH300 C18 column (5 μm, 10×250mm) using a linear gradient of 30-70% acetonitrile/water (0.05% TFA),over 15 min, at a flow rate of 5 mL/min. SQS-1-11-5-18 (19) (5.5 mg, 65%yield) was obtained as a white powder after lyophilization.

Bis(silyl ether) of Echinocystic Acid (S14).

(AFT-I-206) Echinocystic acid S13 (18 mg, 38 μmol, 1.0 equiv) wassuspended in CH₂Cl₂ (10 mL) and cooled in an ice bath. 2,6-lutidine (71μL, 0.61 mmol, 16 equiv) was then added followed by triethylsilyltrifluoromethanesulfonate (69 μL, 0.31 mmol, 8.0 equiv) and the reactionmixture was stirred at 0° C. for 1 h. After this time, the contents werewashed with saturated NaHCO₃ (5 mL) and the aqueous phase was extractedwith CH₂Cl₂ (2×10 mL). The combined organics were dried over Na₂SO₄,filtered, and concentrated. The crude product was purified by silica gelchromatography (hexanes to 9:1 hexanes/EtOAc) to afford S14 (25 mg, 94%yield).

Protected Echinocystic Acid Saponin Azide S19.

(AFT-I-212) A solution of S14 (25 mg, 36 μmol, 1.0 equiv) and imidate 12(50 mg, 45 mol, 1.25 equiv) in CH₂Cl₂ (5 mL) with 40 mg powdered 4 Åmolecular sieves was cooled to −45° C. and boron trifluoridediethyletherate (0.9 μL, 7 μmol, 0.2 equiv) was added. The mixture wasstirred at this temperature for 0.5 h min, quenched with 0.2 mL oftriethylamine and concentrated. Purification of the residue by silicagel chromatography (0.2% triethylamine in benzene to 97:3 benzene/EtOAc)gave S19 (48 mg, 80% yield) as a white solid.

Protected Echinocystic Acid Saponin Amine S31.

(AFT-I-214) To S19 (52 mg, 31 μmol, 1.0 equiv) dissolved intriethylamine (25 mL) was added a freshly prepared solution of phenylselenol (0.94 mmol, 30 equiv) via cannula. The reaction was stirred at38° C. for 8 h, and the solution was then concentrated to afford ayellow-white solid. The crude mixture was purified by silica gelchromatography (9:1 to 4:1 toluene/EtOAc) to afford the amine S31 (42mg, 83% yield) as a glassy solid.

Fully Protected Aminoacyl Echinocystic Acid Saponin S22.

(AFT-I-215) To a clear, colorless solution of6-((t-butoxycarbonyl)-amino)hexanoic acid (14) (44 mg, 0.19 mmol, 11.5equiv) in tetrahydrofuran (2 mL) at 0° C. was added triethylamine (208μL, 1.49 mmol, 90 equiv) followed by ethyl chloroformate (16.0 μL, 0.17mmol, 10.0 equiv). The turbid, white solution was stirred at 0° C. for2.5 h and then added via cannula to amine S31 (27 mg, 16.6 μmol, 1.0equiv) at 0° C. The reaction mixture was stirred at this temperature for1.5 h and then quenched with water (0.2 mL) and concentrated.Purification by silica gel chromatography (9:1 to 5:1 benzene/EtOAc with0.2% triethylamine) afforded S22 (27 mg, 88% yield) as a white glassysolid.

Aminoacyl Echinocystic Acid Saponin S25.

(AFT-I-216) In a 25 mL round-bottom flask containing S22 (24 mg, 13μmol, 1.0 equiv) was added tetrahydrofuran/ethanol (6 mL, 1:1) and 10%(dry basis) palladium on carbon, wet, Degussa type E101 NE/W (138 mg, 65μmol, 5.0 equiv). The reaction was stirred under hydrogen atmosphere(balloon) at 21° C. for 12 h, and then filtered through a 0.45 μm nylonsyringe filter, washed with methanol (3×10 mL), and concentrated.Successful debenzylation is assessed by the disappearance of aromaticresonances by ¹H NMR in CD₃OD. The crude mixture was then dissolved in apre-cooled (0° C.) solution of trifluoroacetic acid (4 mL, TFA/H₂O 3:1)and stirred for 1.25 h in an ice bath. The reaction was evaporated todryness to afford a white solid that was dissolved in 25%acetonitrile/water (12 mL) and purified via RP-HPLC on an XBridge PrepBEH300 C18 column (5 μm, 10×250 mm) using a linear gradient of 30-70%acetonitrile/water (0.05% TFA), over 15 min, at a flow rate of 5 mL/min.The aminoacyl echinocystic acid saponin S25 eluted as a single peak andwas obtained as a white powder (7.0 mg, 53% yield) after lyophilization.

SQS-1-8-5-18 (20).

(AF-I-223) S25 (7.0 mg, 6.8 gmol, 1.0 equiv) was dissolved inN,N′-dimethylformamide (2 mL) in a 25 mL round-bottom flask andtriethylamine (20 μL, 0.14 mmol, 20 equiv) was injected. A solution of 4(11.8 mg, 34 gmol. 5.0 equiv) in N,N′-dimethylformamide (1.5 mL) wasadded dropwise via syringe and the reaction was stirred at 21° C. in thedark. After 3 h, the contents were diluted with 25% acetonitrile/water(9 mL) and purified via RP-HPLC on an XBridge Prep BEH300 C18 column (5μm, 10×250 mm) using a linear gradient of 30-70% acetonitrile/water(0.05% TFA), over 15 min, at a flow rate of 5 mL/min. SQS-1-8-5-18 (20)(6.8 mg, 80% yield) eluted as a single peak and was obtained as a whitepowder after lyophilization.

Gypsogenin (S16).

(AFT-I-218) In a 25 mL roundbottom flask, hederagenin S15 (45 mg, 95gmol, 1.0 equiv) was suspended in CH₂Cl₂ (3.5 mL), and an aqueoussolution (3.5 mL) of 0.5 M NaHCO₃ (147 mg), 0.05 M K₂CO₃ (24.2 mg), andtetrabutylammonium chloride hydrate (28 mg, 95 μmol, 1.0 equiv) was thenadded. To the vigorously stirred mixture, TEMPO (14.8 mg, 95 gmol, 1.0equiv) was added followed by N-chlorosuccinimide (38.0 mg, 0.29 mmol,3.0 equiv) and the reaction was stirred for 2 h in the dark. Thecontents were partitioned in a separation funnel and extracted withCH₂Cl₂ (3×10 mL). The combined organic extracts were dried over Na₂SO₄,filtered, and concentrated to give a crude product that was purified bysilica gel chromatography (hexanes/EtOAc, 7:3) to afford the desiredgypsogenin triterpene S16 (32 mg, 72% yield).

Silyl Ether of Gypsogenin (S17).

(AFT-I-219) A suspension of gypsogenin S16 (32 mg, 68 μmol, 1.0 equiv)in CH₂Cl₂ (10 mL) was cooled in an ice bath and 2,6-lutidine (63 μL,0.54 mmol, 8.0 equiv) and triethylsilyl trifluoromethanesulfonate (62μL, 0.27 mmol, 4.0 equiv) were injected. After stirring for 1 h, thecontents were washed with saturated NaHCO₃ (7 mL) and the aqueous phasewas extracted with CH₂Cl₂ (3×10 mL). The combined organics were dried(Na₂SO₄), filtered, and concentrated. The crude product was purifiedseveral times by silica gel chromatography (hexanes to 4:1hexanes/EtOAc) to afford S17 (26 mg, 65% yield).

Protected Gypsogenin Saponin Azide S20.

(AF-I-224) A solution of S17 (26 mg, 44 μmol, 1.0 equiv) and imidate 12(55 mg, 49 μmol, 1.1 equiv) in CH₂Cl₂ (6 mL) with 40 mg powdered 4 Åmolecular sieves was stirred at 21° C. for 30 min and then cooled to−45° C. before injecting boron trifluoride diethyletherate (1.1 μL, 9μmol, 0.2 equiv). The reaction was stirred at this temperature for 0.5h, quenched with triethylamine (0.2 mL) and concentrated. Purificationby silica gel chromatography (benzene to 97:3 benzene/EtOAc) gavedesired product plus some impure mixture that was furtherchromatographed to afford S20 (48 mg, 70% yield) as a glassy solid.

Protected Gypsogenin Saponin Amine S32.

(AF-I-225) To a solution of S20 (50 mg, 32 μmol, 1.0 equiv) intriethylamine (27 mL) was added a freshly prepared solution of phenylselenol (1.07 mmol, 32 equiv) via cannula. After stirring at 38° C. for8 h, the solution was concentrated to give a yellow-white solid, whichwas purified by silica gel chromatography (9:1 to 8:2 toluene/EtOAc) toafford S32 (35 mg, 72% yield) as a white solid.

Fully Protected Aminoacyl Gypsogenin Saponin S23.

(AFT-I-226) To a solution of 14 (61 mg, 0.27 mmol, 11.5 equiv) intetrahydrofuran (3 mL) at 0° C. was added triethylamine (290 pt, 2.1mmol, 90 equiv) followed by ethyl chloroformate (22 μL, 0.23 mmol, 10equiv), which turned the clear solution turbid white. The acidactivation was allowed to proceed for 2.5 h at 0° C. and the entiresolution was cannula transferred into a schlenck containing amine S32(35 mg, 23 μmol, 1.0 equiv). The reaction mixture was stirred at 0° C.for 1.5 h and then quenched with water (90 μL), at which point thesolution turned from turbid, white to clear. The contents were thenevaporated to dryness and purified by silica gel chromatography (9:1 to5:1 benzene/EtOAc with 0.2% triethylamine) to afford S23 (34 mg, 86%yield) as a white glassy solid.

Aminoacyl Gypsogenin Saponin S26.

(AF-I-227) In a 25 mL round-bottom flask, S23 (27 mg, 15.5 μmol, 1.0equiv) was dissolved in 6 mL tetrahydrofuran/ethanol (1:1) and 10% (drybasis) palladium on carbon, wet, Degussa type E101 NE/W (166 mg, 78μmol, 5 equiv) was added. The reaction was stirred under hydrogenatmosphere (balloon) at 21° C. for 12 h. After this time, the mixturewas filtered through a 0.45 mm nylon syringe filter, washed withmethanol (20 mL) and concentrated. Successful debenzylation was assessedby the disappearance of aromatic resonances by ¹H NMR in CD₃OD. Theresidue was then dissolved in a pre-cooled (0° C.) solution oftrifluoroacetic acid/water (4 mL, 3:1) and stirred for 1.25 h in an icebath. After this time, the reaction was evaporated to dryness to give awhite solid that was purified by RP-HPLC using a 30-70%acetonitrile/water (0.05% TFA) linear gradient, over 15 min, at a flowrate of 5 mL/min. The desired product S26 (13 mg, 82% yield) wasobtained as a white powder after lyophilization.

SQS-1-9-5-18 (21).

(AF-I-230) In a 25 mL round-bottom flask, amine S26 (6.6 mg, 6.5 μmol,1.0 equiv) was dissolved in N,N′-dimethylformamide (2.0 mL) andtriethylamine (18 μL, 0.13 mmol, 20 equiv) was injected. To thissolution, 4 (11.1 mg, 32 μmol, 5.0 equiv) dissolved inN,N′-dimethylformamide (1.5 mL) was added dropwise and the reactionmixture was stirred at 21° C. for 3 h in the dark. After this time, thecontents were diluted with 9 mL 25% acetonitrile/water (0.05% TFA) anddirectly purified by RP-HPLC using a 30-70% acetonitrile/water (0.05%TFA) linear gradient, over 15 min, at a flow rate of 5 mL/min.SQS-1-9-5-18 (21) (4.5 mg, 56% yield) was obtained as a white powderafter lyophilization.

Bis(silyl ether) of Hederagenin (S18).

(AFT-I-228) Hederagenin S15 (35 mg, 74 μmol, 1.0 equiv) was suspended inCH₂Cl₂ (15 mL) and cooled in an ice bath. 2,6-lutidine (138 μL, 1.18mmol, 16 equiv) was then added followed by triethylsilyltrifluoromethanesulfonate (134 μL, 0.59 mmol, 8.0 equiv) and thereaction mixture was stirred at 0° C. for 1 h. After this time, thecontents were washed with saturated NaHCO₃ (10 mL) and the aqueous phasewas extracted with CH₂Cl₂ (2×15 mL). The combined organics were dried(Na₂SO₄), filtered, and concentrated. The crude product was purifiedseveral times by silica gel chromatography (hexanes to 4:1hexanes/EtOAc) to afford S18 (45 mg, 81% yield).

Protected Hederagenin Saponin Azide S21.

(AFT-1-232) A solution of S18 (28 mg, 40 μmol, 1.1 equiv) and imidate 12(41 mg, 36.7 mol, 1.0 equiv) in CH₂Cl₂ (5 mL) with 35 mg powdered 4 Åmolecular sieves was cooled to −45° C. and boron trifluoridediethyletherate (0.9 μL, 7 mol, 0.2 equiv) was added. The mixture wasstirred at this temperature for 0.5 h, quenched with 0.2 mL oftriethylamine and concentrated. Purification of the residue by silicagel chromatography (0.2% triethylamine in benzene to 98:2 benzene/EtOAc)afforded S21 (43 mg, 71% yield) as a glassy solid.

Protected Hederagenin Saponin Amine S33.

(AFT-I-233) To S21 (44 mg, 26.5 μmol, 1.0 equiv) dissolved intriethylamine (24 mL) was added a freshly prepared solution of phenylselenol (0.80 mmol, 30 equiv) via cannula. The reaction was stirred at38° C. for 8 h and the solution was then concentrated to afford ayellow-white solid. The crude mixture was purified by silica gelchromatography (9:1 to 4:1 toluene/EtOAc) to afford the amine S33 (34.5mg, 80% yield) as a glassy solid.

Fully Protected Aminoacyl Hederagenin Saponin S24.

(AFT-I-234) To a solution of 6-((t-butoxycarbonyl)-amino)hexanoic acid(14) (56 mg, 0.24 mmol, 11.5 equiv) in tetrahydrofuran (2.5 mL) at 0° C.was added triethylamine (263 μL, 1.89 mmol, 90 equiv) followed by ethylchloroformate (20.0 μL, 0.21 mmol, 10.0 equiv). The turbid, whitesolution was stirred at 0° C. for 2.5 h and then added via cannula toamine S33 (34.5 mg, 21.0 μmol, 1.0 equiv) at 0° C. The reaction mixturewas stirred at this temperature for 1.5 h and then quenched with water(0.2 mL) and concentrated. Purification by silica gel chromatography(9:1 to 5:1 benzene/EtOAc with 0.2% triethylamine) afforded S24 (36.5mg, 92% yield) as a white solid.

Aminoacyl Hederagenin Saponin S27.

(AFT-I-235) In a 25 mL round-bottom flask, S24 (30 mg, 16.3 gmol, 1.0equiv) was dissolved in tetrahydrofuran/ethanol (7 mL, 1:1) and 10% (drybasis) palladium on carbon, wet, Degussa type E101 NE/W (173 mg, 81gmol, 5.0 equiv) was added. The reaction was stirred under hydrogenatmosphere (balloon) at 21° C. for 12 h, and then filtered through a0.45 μm nylon syringe filter, washed with methanol (3×10 mL) andconcentrated. Successful debenzylation is assessed by the disappearanceof aromatic resonances by ¹H NMR in CD₃OD. The crude mixture was thendissolved in a pre-cooled (0° C.) solution of trifluoroacetic acid (4mL, TFA/H₂O 3:1) and stirred for 1.25 h in an ice bath. The reaction wasevaporated to dryness and the white solid was dissolved in 30%acetonitrile/water (0.05% TFA) (14 mL) and purified via RP-HPLC on anXBridge Prep BEH300 C18 column (5 μm, 10×250 mm) using a linear gradientof 30-70% acetonitrile/water (0.05% TFA), over 15 min, at a flow rate of5 mL/min. The desired product S27 was obtained as a white powder (11.0mg, 66% yield) after lyophilization.

SQS-1-10-5-18 (22).

(AF-I-236) S27 (6.0 mg, 5.8 μmol, 1.0 equiv) was dissolved inN,N′-dimethylformamide (2.5 mL) in a 25 mL round-bottom flask andtriethylamine (16.3 μL, 0.12 mmol, 20 equiv) was injected. A solution of4 (10.1 mg, 29 μmol, 5.0 equiv) in N,N′-dimethylformamide (1.5 mL) wasadded dropwise via syringe and the reaction was stirred at 21° C. for 3h. After this time, the contents were diluted with 25%acetonitrile/water (9 mL) and purified via RP-HPLC on an XBridge PrepBEH300 C18 column (5 μm, 10×250 mm) using a linear gradient of 30-70%acetonitrile/water (0.05% TFA), over 15 min, at a flow rate of 5 mL/min.SQS-1-10-5-18 (22) (4.2 mg, 57% yield) was obtained as a white powderafter lyophilization.

In summary, extensive structure-function studies of novel iodinatedsaponins based on QS-21 have identified echinocystic acid derivative 20(SQS-1-8-5-18) as a minimal saponin immunoadjuvant with potent activityand dramatically reduced toxicity compared to the natural product (FIG.5), as well as improved synthetic accessibility relative to previouslyreported variants. Subtyping of the IgG antibodies elicited byechinocystic acid derivative 20, as well as the closely-related quillaicacid derivative 16 (SQS-1-0-5-18) and caulophyllogenin derivative 19(SQS-1-11-5-18), indicates that IgG1 and IgG2b subclasses predominate.The mouse IgG1 subclass is associated with Th2 cell responses (humoralimmunity), whereas the IgG2b, together with the IgG2a, are related toTh1 responses (cellular immunity) and are known to induce potentimmunotherapeutic effector functions, including complement-dependentcytotoxicity and antibody-dependent cellular toxicity. Similar resultswere obtained with SQS-21. Thus, despite the considerable structuraldifferences between these truncated saponins and QS 21, they elicit bothTh1 and Th2 immunity, a hallmark of QS 21 itself.

To date, investigation of structural requirements within the triterpenedomain of QS 21 has been hampered by the challenges associated withchemoselective modification of the natural product and by materialthroughput limitations for synthetic analogues that incorporatealternative triterpenes. Thus, the discovery that the entire branchedtrisaccharide domain can be omitted while retaining potent adjuvantactivity and attenuating toxicity has opened the door to investigationof such triterpene modifications by semisynthesis from alternative,readily available triterpene precursors.

These studies revealed that the C4-aldehyde substituent is dispensablefor potent adjuvant activity while the C16-hydroxyl group enhancesactivity in these truncated saponins. In contrast, the C4-aldehydesubstituent of QS 21 has been suggested previously to react with aminogroups on T cell surface receptors through Schiff base formation,providing co-stimulation necessary for T-cell activation and Th1cellular immunity. This hypothesis was based on the finding thatreductive amination of the C4-aldehyde substituent of QS 21 providesamine derivatives with significantly attenuated adjuvant activity.However, this modification not only removes the C4-aldehyde substituentbut also introduces a positively-charged amino group at this position,which may alternatively compromise non-covalent interactions with aputative receptor or otherwise interfere with proper biodistribution orsubcellular localization of the adjuvant. Along similar lines, QS-21variant 2 (SQS-0-0-5-11), which retains the C4-aldehyde substituent butcarries a positively-charged amino functionality in the acyl chaindomain, was shown to be likewise inactive. While it remains possiblethat QS 21 and these modified, synthetic variants may have distinctmolecular targets, they appear to induce similar cellular effects invivo.

Conversely, the finding disclosed herein that the C16-hydroxyl groupenhances adjuvant activity in these truncated saponins suggests apreviously unappreciated role for this functionality, perhaps instabilizing saponin conformation and/or interacting directly with aputative receptor. These results are consistent with reports of otheradjuvant-active saponins that possess the C16-hydroxyl group but lackthe triterpene C4-aldehyde substituent.

1. A compound of formula (I), or a pharmaceutically acceptable salt thereof,

wherein

is a single or double bond; W is C(O)R, CH₂OR or CH₂R, wherein R is H, or an optionally substituted group selected from acyl, arylalkyl, aryl, heteroaryl, aliphatic, heteroaliphatic, cycloaliphatic and heterocyclyl groups; V is H or OH; Y is O; Z is a linear oligosaccharide or an optionally substituted group selected from the group consisting of amine, amide, acyl, arylalkyl, aryl, heteroaryl, aliphatic, heteroaliphatic, cycloaliphatic and heterocyclyl groups.
 2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein Z is a linear tetrasaccharide or a linear trisacchride with an acyl chain attached to a first sugar residue of the linear tetrasaccharide or a linear trisacchride, wherein the first sugar residue is the sugar residue that is attached directly to Y.
 3. The compound of claim 2, or a pharmaceutically acceptable salt thereof, wherein R is H.
 4. The compound of claim 3, or a pharmaceutically acceptable salt thereof, wherein W is Me and V is OH.
 5. The compound of claim 3, or a pharmaceutically acceptable salt thereof, wherein W is CHO and V is OH.
 6. The compound of claim 3, or a pharmaceutically acceptable salt thereof, W is CH₂OH and V is OH.
 7. The compound of claim 2, or a pharmaceutically acceptable salt thereof, wherein the acyl chain attached to the first sugar residue is 6-(4-iodobenzoylamino)-hexanoyl.
 8. A compound of formula (II), or a pharmaceutically acceptable salt thereof,

or a pharmaceutically acceptable salt thereof, wherein W is C(O)R, CH₂OR or CH₂R; wherein R is H, or an optionally substituted group selected from acyl, arylalkyl, aryl, heteroaryl, aliphatic, heteroaliphatic, cycloaliphatic and heterocyclyl groups; V is H or OH, X is CH₂R_(m), C(O)R_(m), CH₂OR_(m), CH₂R_(m), OR_(m), or NHR_(m), wherein R_(m) is H, or an optionally substituted group selected from acyl, arylalkyl, aryl, heteroaryl, aliphatic, heteroaliphatic, cycloaliphatic and heterocyclyl groups, and each occurrence of R_(n) is independently a hydrogen, a monosaccharide, a disaccharide or a trisaccharide.
 9. The compound of claim 8, or a pharmaceutically acceptable salt thereof, wherein R is H.
 10. The compound of claim 9, or a pharmaceutically acceptable salt thereof, wherein W is Me and V is OH.
 11. The compound of claim 9, or a pharmaceutically acceptable salt thereof, wherein W is CHO and V is OH.
 12. The compound of claim 9, or a pharmaceutically acceptable salt thereof, W is CH₂OH and V is OH.
 13. The compound of any one of claim 8, or a pharmaceutically acceptable salt thereof, wherein X is NHR_(m) and wherein R_(m) is 6-(4-iodobenzoylamino)-hexanoyl.
 14. A pharmaceutical composition, comprising: the compound of claim 1, or a pharmaceutically acceptable salt thereof; and an immunologically effective amount of an antigen.
 15. A method for immunizing a subject, comprising: administering to the subject the pharmaceutical composition of claim
 14. 16. A pharmaceutical composition, comprising: the compound of claim 1, or a pharmaceutically acceptable salt thereof; and an effective amount of a cytotoxic drug.
 17. A method for enhancing the effect of a cytotoxic drug in a subject, comprising: administering to the subject the pharmaceutical composition of claim
 16. 18. A compound of formula (III),

wherein W is Me, —CHO, or —CH₂OH, and V is H or OH.
 19. The use of the compound of claim 18 as an intermediate in the synthesis of a minimal saponin analogue.
 20. A process for preparing the compound of claim 18, comprising the steps of: a) reacting a compound of formula (100) with a protecting group to form a compound of formula (101), wherein W is Me, CHO, CH₂OH, or CH₂OR_(p); wherein R_(p) is H or a suitable protecting group as necessary to achieve regioselectivity; V is H or OR_(p), and TES is a triethylsilyl protecting group;

b) reacting the compound of formula (101) with the compound of formula (102) to form the compound of formula (103), wherein Bn is a benzyl protecting group;

c) reacting the compound of formula (103) with a reducing agent to form the compound of formula (104);

d) coupling the compound of formula (104) with compound of formula (105) in the presence of an activating agent to form the compound of formula (106), wherein Boc is a tert-butyloxycarbonyl protecting group;

e) deprotecting the compound of formula (106) to form the compound of formula (III). 